Methods for treating an ischemic disorder and improving stroke outcome

ABSTRACT

The present invention provides for a method of treating an ischemic disorder in a subject which comprises administering to the subject a pharmaceutically acceptable form of inactivated Factor IX in a sufficient amount over a sufficient period of time to inhibit coagulation so as to treat the ischemic disorder in the subject.

This application is a §371 of PCT International Application No.PCT/US97/17229, filed Sep. 25, 1997, designating the United States ofAmerica, which was a continuation-in-part and claimed priority of U.S.Ser. No. 08/721,447 filed Sep. 27, 1996 now abandoned the content ofwhich is hereby incorporated by reference in their entireties into thepresent application.

The invention disclosed herein was made with Government support underNational Institutes of Health, National Heart, Lung and Blood Instituteaward HL55397 of the Department of Health and Human Services.Accordingly, the U.S. Government has certain rights in this invention.

Throughout the application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art as known to those skilled therein as ofthe date of the invention described and claimed herein.

BACKGROUND OF THE INVENTION

Treatment of ischemic disorders has been the focus of research for manyyears. The recent availability of transgenic mice has led to aburgeoning number of reports describing the effects of specific geneproducts on the pathophysiology of stroke. Although focal cerebralischemia models in rats have been well-described, descriptions of amurine model of middle cerebral artery occlusion are scant, and sourcesof potential experimental variability remain undefined.

Acute neutrophil recruitment to postischemic cardiac or pulmonary tissuehas deleterious effects in the early reperfusion period, but themechanisms and effects of neutrophil influx in the pathogenesis ofevolving stroke remains controversial.

SUMMARY OF THE INVENTION

The present invention provides for a method for treating an ischemicdisorder in a subject which comprises administering to the subject apharmaceutically acceptable form of a selectin antagonist in asufficient amount over a sufficient time period to prevent white bloodcell accumulation so as to treat the ischemic disorder in the subject.The invention further provides a method for treating an ischemicdisorder in a subject which comprises administering to the subjectcarbon monoxide gas in a sufficient amount over a sufficient period oftime thereby treating the ischemic disorder in the subject. Theinvention further provides a method for treating an ischemic disorder ina subject which comprises administering to the subject apharmaceutically acceptable form of inactivated Factor IX in asufficient amount over a sufficient period of time to inhibitcoagulation so as to treat the ischemic disorder in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Neutrophil accumulation following focal cerebral ischemia andreperfusion in the mouse. Right middle cerebral artery occlusion wasperformed for 45 minutes, followed by 23 hours of reperfusion in maleC57B1/J6 mice. One hour prior to middle cerebral artery occlusion,≈3.3×10⁵ ¹¹¹ In-labeled neutrophils were injected into the tail vein.Ipsilateral (right hemispheric) and contralateral (left hemispheric)counts were obtained and normalized per gm of tissue. (n=7, **=p<0.01).

FIGS. 2A, 2B, 2C and 2D. Effect of preoperative neutrophil depletion onindices of stroke outcome. C57B1/J6 male mice were subjected totransient middle cerebral artery occlusion as described above (WildType, n=16), and compared with a similar procedure performed in miceimmunodepleted of neutrophils during the three days prior to the day ofsurgery (PMN -, n=18). FIG. 2A. Infarct volumes, calculated based on TTCstained serial cerebral sections, and expressed as the % ipsilateralhemispheric volume. FIG. 2B. Neurologic deficit score, graded prior toanesthesia 24 hours following transient middle cerebral arteryocclusion; 4 represents the most severe neurologic deficit. FIG. 2C.Cerebral blood flow, measured by laser doppler flow measurements 2 mmposterior to the bregma, expressed as % contralateral hemispheric bloodflow. FIG. 2D. Mortality at 24 hours following transient middle cerebralartery occlusion. (*=p<0.05, **=p<0.01, ***=p<0.001).

FIG. 3. Expression of Intercellular Adhesion Molecule-1 (ICAM-1)transcripts 24 hours following middle cerebral artery occlusion. RNA wasprepared from the ipsilateral (infarct) and the contralateral(noninfarct) hemispheres from the same mouse, and an agarose gel wasloaded with 20 μg of total RNA per lane. After overnight transfer to anylon membrane, the Northern blot was probed with a ³²P-labeled 1.90 kbmurine ICAM-1 cDNA³³. A β-actin probe was used for a control.

FIGS. 4A and 4B. Expression of Intercellular Adhesion Molecule-1(ICAM-1) antigen in the cerebral microvasculature 24 hours followingmiddle cerebral artery occlusion. A coronal section of brain wasobtained for ICAM-1 immunostaining, so that the noninfarcted andinfarcted hemispheres from the same brain could be compared underidentical staining conditions. Staining was performed using a ratanti-murine ICAM-1 antibody, with sites of primary antibody bindingvisualized by alkaline phosphatase. FIG. 4A. Cerebral microvessel is thecontralateral (noninfarcted) section of a brain obtained 24 hours aftermiddle cerebral artery occlusion. FIG. 4B. Cerebral microvessel from theipsilateral (infarcted) hemisphere from the same section of brain asshown in FIG. 4A. Endothelial cells from ipsilateral cerebralmicrovessels demonstrate increased expression of ICAM-1 (bright redstaining). Magnification 250×.

FIGS. 5A and 5B. Cerebrovascular anatomy in homozygous null ICAM-1 mice(FIG. 5B) and wild type controls (FIG. 5A). India ink staining ofcerebrovascular anatomy with an inferior view of the Circle of Willisdemonstrates that there were no gross anatomic differences in thevascular pattern of the cerebral circulation, with intact posteriorcommunicating arteries in both.

FIGS. 6A and 6B. TTC-stained serial sections at 24 hours fromrepresentative wild type (FIG. 6A) or homozygous null ICAM-1 mice (FIG.6B) subjected to transient middle cerebral artery occlusion. The palewhite area in the middle cerebral artery territory represents infarctedbrain tissue, whereas viable tissue stains brick red. Quantification ofinfarct volumes by planimetry of serial cerebral sections in multipleexperiments is shown in FIG. 7A.

FIGS. 7A, 7B, 7C and 7D. Role of ICAM-1 in stroke outcome. Transientmiddle cerebral artery occlusion was performed as described in ICAM-1+/+ (Wild Type, n=16) or ICAM-1 −/− (n=13) mice, and indices of strokeoutcome measured as described in FIG. 2. FIG. 7A. Effect of ICAM-1 oninfarct volume, FIG. 7B. neurologic deficit score, FIG. 7C. cerebralblood flow, and FIG. 7D, mortality. (*=p<0.05, **=p<0.01).

FIGS. 8A and 8B. Effect of hypoxia on Weibel-Palade body exocytosis.FIG. 8A. Human umbilical veins were exposed to hypoxia (pO₂ 15-20 Torr)or normoxia for the indicated durations, and vonWillebrand factor (vWF),secretion quantified by ELISA. ***=p<0.001 for hypoxia vs normoxia. FIG.8B. Similar experiments were performed for 8 hrs in the presence of 2 mMCa⁺⁺ (Ca⁺⁺ 2 mM), 0 mM Ca⁺⁺ (Ca⁺⁺-free), or 0 mM Ca⁺⁺with 2 mM EGTAadded to chelate residual extracellular Ca^(++ (Ca) ⁺⁺-free+EGTA).

FIGS. 9A, 9B, 9C and 9D. Effect of endothelial hypoxia on P-selectinexpression and neutrophil adhesion. FIG. 9A. P-selectin expression onHUVECs exposed to normoxia or hypoxia, determined by specific binding ofradiolabelled monoclonal anti-P-selectin IgG (WAPS12.2 clone). Data areexpressed as relative binding compared with the 4 hr normoxic timepoint. FIG. 9B. Effect of inhibiting protein synthesis onhypoxia-induced P-selectin expression. In a separate experiment, theeffect of cyloheximide (10 μg/mL,+CHX) added at the start of the 4 hournormoxic or hypoxic period on P-selectin expression is shown. Comparisonis made to simultaneous experiments performed in the absence ofcyloheximide (−CHX), with data expressed as relative binding comparedwith normoxic (−CHX) binding. Means±SEM are shown; *=p<0.05 vs normoxia(−CHX); †=p<0.05 vs normoxia (+CHX). Inset: Effect of cyloheximide (10μg/mL) on protein synthesis at 4 hrs, measured as trichloroaceticacid-precipitable ³⁵S-labeled proteins following ³⁵S-methionine and³⁵S-cysteine administration. FIG. 9C. ¹¹¹Indium-labeled neutrophilbinding to normoxic (N) or hypoxic (H) human umbilical vein endothelialmonolayers at 4 hrs, in the presence of a blocking anti-P-selectinantibody (WAPS 12.2 clone) or a nonblocking anti-P-selectin antibody(AC1.2 clone). Means±SEM are shown; **=p<0.01.

FIGS. 10A, 10B, and 10C. Role of neutrophils and endothelial P-selectinin rodent cardiac preservation followed by heterotopic transplantation.FIG. 10A. Rat cardiac preservation. Hearts were transplanted immediatelyafter harvest (Fresh, n=8) or preserved for 16 hrs in lactated Ringer'ssolution at 4° C. followed by transplantation (Prsvd, n=4). The effectof administering non-blocking anti-P-selectin antibody (AC1.2, n=3),immunodepleting recipients of neutrophils prior to donor heartimplantation (−PMN, n=4), or administering 250 μg of blockinganti-P-selectin IgG (n=4) 10 minutes prior to reperfusion on cardiacgraft survival (shaded bars) and leukostasis (myeloperoxidase activity,dark bars). Means±SEM are shown; For graft survival, c vs a, p<0.0001; gvs c, p<0.05; i vs e or c, p<0.05. For graft neutrophil infiltration, dvs b, p<0.01; h vs d, p<0.05; j vs d or f, p<0.05. FIG. 10B. Role ofcoronary endothelial P-selectin in cardiac preservation, using donorhearts from P-selectin null (or wild type control) mice that wereflushed free of blood prior to preservation. Graft survival was assessedby the presence/absence of cardiac electrical/mechanical activityexactly ten minutes following reestablishment of blood flow. FIG. 10C:Quantification of neutrophil infiltration by measurement ofmyeloperoxidase activity (dABs-460 nm/min) as described^(15,18). (Forbars shown from left to right, n=14, 8,13, and 7, respectively with Pvalues indicated).

FIGS. 11A and 11B. Weibel-Palade body release during human cardiacsurgery in 32 patients. FIG. 11A. Coronary sinus blood was sampled atthe start (CS₁) and conclusion (CS₂) of the ischemic period (aorticcross-clamping). ELISAs were performed for thrombomodulin (TM) and vWF.FIG. 11B. vWF immunoelectrophoresis of a representative sample of CS₁and CS₂ blood from the same patient (dilution factors are indicated).There is an increase in high molecular weight multimers detected in theCS₂ samples.

FIGS. 12A, 12B, 12C and 12D. Overview of operative setup for murinefocal cerebral ischemia model. FIG. 12A. Suture based retraction systemis shown in the diagram. FIG. 12B. View through the operatingmicroscope. The large vascular stump represents the external carotidartery, which is situated inferomedially in the operating field. FIG.12C. Photograph of heat-blunted occluding suture of the indicated gauge(5-0 [bottom] or 6-0 nylon [top]). FIG. 12D. Schematic diagram of murinecerebrovascular anatomy, with thread in the anterior cerebral artery,occluding the middle cerebral artery at its point of origin.

FIG. 13. Comparison of cerebrovascular anatomy between strains of mice.Following anesthesia, mice were given an intracardiac injection of Indiaink followed by humane euthanasia. An intact Circle of Willis can beobserved in all strains, including bilateral posterior communicatingarteries, indicating that there are no gross strain-related differencesin cerebrovascular anatomy.

FIGS. 14A, 14B and 14C. Effects of mouse strain on stroke outcome. Mice(20-23 gm males) were subjected to 45 minutes of MCA occlusion (using 12mm 6.0 occluding suture) followed by 24 hours of reperfusion, andindices of stroke outcome determined. FIG. 14A. Effects of strain oninfarct volume, determined as a percentage of ipsilateral hemisphericvolume, as described in the Methods section. FIG. 14B. Effects of strainon neurological deficit score, graded from no neurologic deficit (0) tosevere neurologic deficit (4), with scores determined as described inthe Methods section. FIG. 14C. Effects of strain on cerebral blood flow,measured by laser doppler flowmetry as relative flow over the infarctedterritory compared with blood flow over the contralateral (noninfarcted)cortex. Strains included 129J (n=9), CD1 (n=11), and C57/B16 mice(n=11); *=p<0.05 vs 129J mice.

FIGS. 15A, 15B and 15C. Effects of animal size and diameter of theoccluding suture on stroke outcome. Male CD-1 mice of the indicatedsizes were subjected to middle cerebral artery occlusion (45 minutes)followed by reperfusion (24 hours) as described in the Methods section.Suture size (gauge) is indicated in each panel. Small animals (n=11)were those between 20-25 gm (mean 23 gm), and large animals were between28-35 gm (mean 32 gm, n=14 for 6.0 suture, n=9 for 5.0 suture). FIG.15A. Effects of animal/suture since on infarct volume, FIG. 15B.neurological deficit score, and FIG. 15C. cerebral blood flow, measuredas described in FIG. 14. P values are as shown.

FIGS. 16A, 16B and 16C. Effects of temperature on stroke outcome. MaleC57/B16 mice were subjected to 45 minutes of MCA occlusion (6.0 suture)followed by reperfusion. Core temperatures were maintained for 90minutes at 37° C. (normothermia, n=11) using an intrarectal probe with athermocouple controlled heating device. In the second group(hypothermia, n=12), animals were placed in cages left at roomtemperature after an initial 10 minutes of normothermia (mean coretemperature 31° C. at 90 minutes). In both groups, after this 90 minuteobservation period, animals were returned to their cages with ambienttemperature maintained at 37° C. for the duration of observation.Twenty-four hours following MCA occlusion, indices of stroke outcomewere recorded; FIG. 16A. infarct volume, FIG. 16B. neurological deficitscore, and FIG. 16C. cerebral blood flow, measured as described in FIG.3. *=p<0.05 values are as shown.

FIGS. 17A, 17B and 17C. Outcome comparisons between permanent focalcerebral ischemia and transient focal cerebral ischemia followed byreperfusion. The MCA was either occluded permanently (n=11) ortransiently (45 minutes, n=17) with 6.0 gauge suture in 22 gram MaleC57/B16 mice, as described in the Methods section. Twenty-four hoursfollowing MCA occlusion, indices of stroke outcome were recorded; FIG.17A. infarct volume, FIG. 17B. neurological deficit score, and FIG. 17C.cerebral blood flow, measured as described in FIG. 14.

FIGS. 18A and 18B. P-selectin expression and neutrophil (PMN)accumulation following middle cerebral artery occlusion (MCAO) in mice.FIG. 18A. P-selectin expression following MCAO and reperfusion. Relativeexpression of P-selectin antigen in the ipsilateral cerebral hemispherefollowing middle cerebral artery occlusion was demonstrated using eithera ¹²⁵I-labeled rat monoclonal anti-P-selectin IgG or a ¹²⁵I-labelednonimmune rat IgG to control for nonspecific extravasation. Experimentswere performed as described in the legend to FIG. 18. Values areexpressed as ipsilateral cpm/contralateral cpm. n=6 for each group,except for control 30 min (n=4); ^(‡)=p<0.001, 30 min reperfusion vsimmediate pre-occlusion; *=p<0.025, change in P-selectin accumulation vschange in control IgG accumulation. FIG. 18B. Time course of PMNaccumulation following focal cerebral ischemia and reperfusion in themouse. For these experiments, ≈3.3×10⁵ ¹¹¹ In-labeled PANS were injectedintravenously into PS wild type (PS +/+) mice 15 minutes prior to middlecerebral artery occlusion (MCAO). ¹¹¹In-PMN accumulation was measuredimmediately following sacrifice as the ratio ofipsilateral/contralateral cpm under the following experimentalconditions: prior to MCAO (Pre-O, n=4), immediately following MCAO(Post-O, n=6), and 10 minutes following MCAO but still prior toreperfusion (:10 Post-O, n=6). To establish the effect of reperfusion onPMN accumulation, reperfusion was initiated following 45 minutes ofischemia. PMN accumulation was measured following 30 minutes (n=6), 300minutes (n=3), and 22 hours (n=8) of reperfusion. Under identicalconditions, PMN accumulation was measured in P-selectin null (PS −/−)mice after 45 minutes of ischemia and 22 hours of reperfusion (n=7,*=p<0.05 vs 45 min MCAO/22 hrs reperfusion in PS +/+ animals).

FIG. 19. Role of P-selectin in the cerebrovascular no-reflow. Cerebralblood flow was measured in PS +/+ (top panel) and PS −/− (middle panel)mice using a laser doppler flow probe, and expressed as the percentageof contralateral (nonischemic) hemispheric blood flow (±SEM). Blood flowwas measured at the following time points: a, prior to MCAO (PS +/+,n=16; PS −/−, n=23); b, immediately following MCAO (PS +/+, n=42; PS−/−, n=40); c, 10 minutes following MCAO but still prior to reperfusion(PS +/+, n=36; PS −/−, n=34); d, immediately following reperfusion (PS+/+, n=36; PS −/−, n=34); e, 30 minutes following reperfusion (PS +/+,n=8; PS −/−, n=5); and f, 22 hours following reperfusion (PS +/+, n=15;PS −/−, n=5). The bottom panel represents an overlay of the top twopanels, with error bars omitted for clarity.

FIG. 20. Cerebrovascular anatomy in homozygous null P-selectin mice, PS−/− (right) and wild type controls, PS +/+ (left). India ink/carbonblack staining of cerebrovascular anatomy with an inferior view of theCircle of Willis demonstrates that there were no gross anatomicdifferences in the vascular pattern of the cerebral circulation, withintact posterior communicating arteries in both.

FIGS. 21A, 21B and 21C. Effect of the P-selectin gene on strokeoutcomes. Middle cerebral artery occlusion was performed for 45 minutes,followed by 22 hours of reperfusion in P-selectin +/+ (n=10) orP-selectin −/− (n=7) mice. Effect of P-selectin on: FIG. 21A. infarctvolume, as evidenced by 2% 2,3,5, triphenyl, 2H-tetrazolium chloride(TTC) staining, and calculated as percent of ipsilateral hemisphere;FIG. 21B. neurologic deficit score, (1=normal spontaneous movements;2=clockwise circling; 3=clockwise spinning; 4=unresponsiveness tonoxious stimuli); FIG. 21C. percent survival at time of sacrifice.(*=p<0.05).

FIGS. 22A, 22B, 22C and 22D. Effect of P-selectin blockade on strokeoutcomes. PS +/+ mice were given either a blocking rat anti-mouseanti-P-selectin IgG (clone RB 40.34, 30 μg/mouse) or a similar dose ofnonimmune rat IgG immediately prior to surgery. FIG. 22A. Cerebral bloodflow at thirty minutes following reperfusion; After 22 hours ofreperfusion, infarct volumes FIG. 22B., neurological deficit scores FIG.22C., and mortality FIG. 22D. are shown. (n=7 for each group, *=p<0.05).

FIGS. 23A and 23B. FIG. 23A. The effect of carbon monoxide inhalation oncerebral infarct volumes. Mice were placed in bell jars, in which theywere exposed to 0.1% CO for 12 hours. After this treatment, they wereremoved from the bell jars and subjected to intraluminal occlusion ofthe middle cerebral artery. At 24 hours, animals were sacrificed andinfarct volumes measured by triphenyltetrazolium chloride (TTC) stainingas shown in FIG. 25. Quantification of infarction volumes (mean ±SEM) isexpressed as the percent of infarction of the ipsilateral hemisphere.These data show that inhaled CO reduces infarct volumes followingstroke.

FIG. 23B. The effect of carbon monoxide inhalation on mortalityfollowing stroke. Experiments were performed as described above.Mortality at 4 hours is shown. These data show that inhaled CO reducesmortality following stroke.

FIGS. 24A, 24B, and 24C. FIG. 24A. Dose-response of inhaled carbonmonoxide on stroke outcome. Experiments are described above. CO wasinhaled at the indicated doses. These data show that inhaled CO reducesinfarct volume in a dose-dependent fashion, with 0.1% providing optimalprotection. FIGS. 24B and 24C. Role of heme oxygenase, the enzyme whichmakes CO, in stroke. Animals were given either vehicle (DMSO) alone as acontrol or zinc protoporphyrin IX (ZnPP) or tin protoporphyrin IX(SnPP). In a final group, mice were given biliverdin (Bili), a compoundwhich is formed along with CO during the process of heme degradation byheme oxygenase. Left panel shows infarction volumes. Right panel showsmortality. These experiments demonstrate that when heme oxygenaseactivity is blocked, stroke outcomes are worse (larger infarcts andhigher mortalities). Because biliverdin administration is notprotective, these data suggest that the other coproduct of hemeoxygenase activity (CO) is protective.

FIG. 25. TTC staining of serial cerebral sections for the animals ofFIG. 23. Infarcted tissue appears white, and viable tissue appears brickred.

FIGS. 26A-26F. Effect of focal cerebral ischemia on heme oxygenase I(HO-I) induction. FIGS. 26A-26C show in situ hybridization of HO-I mRNAin stroke (FIG. 26B) and in controls (FIGS. 26A and 26C). FIGS. 26D-26Fshow immunohistochemistry of HO-I protein. FIG. 26E shows that theprotein is expressed in blood vessels and astrocytes following stroke.FIGS. 26D and 26F show that the protein is not expressed in bloodvessels and astrocytes in controls.

FIG. 27. Effect of focal cerebral ischemia on heme oxygenase I (HO-I)mRNA induction. Contralateral indicates the nonstroke side of the brain.Ipsilateral indicates the brain side subjected to stroke. In bothanimals, the side of the brain subjected to stroke demonstratesincreased HO-I but the nonstroke side does not.

FIG. 28. Effect of hypoxia on heme oxygenase I (HO) induction. Miceexposed to a hypoxic environment for 12 hours (to simulate ischemia)show an increase in heme oxygenase I mRNA compared with normoxiccontrols. These data show a potential mechanism whereby hypoxicpre-exposure can also confer protection against subsequent ischemicevents, which was found to be true in mice subjected to hypoxia followedby stroke.

FIG. 29. Effect of hypoxia on heme oxygenase I (HO-I) protein expressionin mouse brain endothelial cells. Hypoxia causes HO-I protein levels toincrease in these brain-derived endothelial cells.

FIG. 30. Effect of hypoxia on heme oxygenase I (HO-I) mRNA induction inmouse brain endothelial cells. Hypoxia causes HO-I mRNA levels toincrease in these brain-derived endothelial cells.

FIGS. 31A-31D. P-selectin expression and neutrophil (PMN) accumulationfollowing middle cerebral artery occlusion (MCAO) in mice. FIG. 31A.P-selectin expression following MCAO and reperfusion. Relativeexpression of P-selectin antigen in the ipsilateral cerebral hemispherefollowing middle cerebral artery occlusion was demonstrated using eithera ¹²⁵I-labelled rat monoclonal anti-P-selectin IgG or a ¹²⁵I-labellednonimmune rat IgG to control for nonspecific extravasation. Values areexpressed as ipsilateral cpm/contralateral cpm. n=6 for each group,except for control 30 min (n=4); ^(‡)=p<0.001, 30 min reperfusion vsimmediate pre-occlusion; *=p<0.025, change in P-selectin accumulation vschange in control IgG accumulation. FIGS. 31B and 31C.Immunohistochemical localization of P-selectin expression in a sectionof brain from a mouse subjected to 45 minutes of MCAO followed by 1 hourof reperfusion. Ipsilateral and contralateral cerebral cortical sectionsare shown from the same mouse. Arrows point to a cerebral microvessel,with dark brown color representing P-selectin expression at theendothelial cell surface. FIG. 31D. Time course of PMN accumulationfollowing focal cerebral ischemia and reperfusion in the mouse. Forthese experiments, ≈3.3×10⁵ ¹¹¹In-labelled PMNs were injectedintravenously into PS wild type (PS +/+) mice 15 minutes prior to middlecerebral artery occlusion (MCAO). ¹¹¹In-PMN accumulation was measuredimmediately following sacrifice as the ratio ofipsilateral/contralateral cpm under the following experimentalconditions: prior to MCAO (Pre-O, n=4), immediately following MCAO(Post-O, n=6), and 10 minutes following MCAO but still prior toreperfusion (:10 Post-O, n=6). To establish the effect of reperfusion onPMN accumulation, reperfusion was initiated following 45 minutes ofischemia. PMN accumulation was measured following 30 minutes (n=6), 300minutes (n=3), and 22 hours (n=8) of reperfusion. Under identicalconditions, PMN accumulation was measured in P-selectin null (PS −/−)mice after 45 minutes of ischemia and 22 hours of reperfusion (n=7,*=p<0.05 vs 45 min MCAO/22 hrs reperfusion in PS +/+ animals).

FIGS. 32A-32C. Role of P-selectin in the cerebrovascular no-reflow.Cerebral blood flow was measured in PS +/+ (top panel) and PS −/−(middle panel) mice using a laser doppler flow probe, and expressed asthe percentage of contralateral (nonischemic) hemispheric blood flow(±SEM). Blood flow was measured at the following time points: a, priorto MCAO (PS +/+, n=16; PS −/−, n=23); b, immediately following MCAO (PS+/+, n=42; PS −/−, n=40); c, 10 minutes following MCAO but still priorto reperfusion (PS +/+, n=36; PS −/−, n=34); d, immediately followingreperfusion (PS +/+, n=36; PS −/−, n=34); e, 30 minutes followingreperfusion (PS +/+, n=8; PS −/−, n=5); and f, 22 hours followingreperfusion (PS +/+, n=15; PS −/−, n=5). The bottom panel represents anoverlay of the top two panels, with error bars omitted for clarity.

FIGS. 33A-33B. Effect of the P-selectin gene on stroke outcomes. Middlecerebral artery occlusion was performed for 45 minutes, followed by 22hours of reperfusion in P-selectin +/+ (n=10) or P-selectin −/31 (n=7)mice. Effect of P-selectin on: FIG. 33A. infarct volume, as evidenced by2% 2,3,5, triphenyl, 2H-tetrazolium chloride (TTC) staining, andcalculated as percent of ipsilateral hemisphere; FIG. 33B. percentsurvival at time of sacrifice. Means±SEM are indicated, within thenumbers of animals from which the percentage survival was calculatedindicated above the survival bars (*=p<0.05).

FIG. 34. Effect of P-selectin blockade on stroke outcomes. PS +/+ micewere given either a blocking rat anti-mouse anti-P-selectin IgG (cloneRB 40.34, 30 μg/mouse) or a similar dose of nonimmune rat IgGimmediately prior to middle cerebral artery occlusion (Pre-MCAO; n=7 foreach group) or after occlusion of the middle cerebral artery (Post-MCAO;n=9 for the control antibody, n=6 for the functionally blockinganti-P-selectin antibody). In both cases, the intraluminal occludingsuture was withdrawn after a 45 minute ischemic period to simulateclinical reperfusion. After 22 hours of reperfusion, infarct volumes(dark bars), relative cerebral blood flow at thirty minutes followingreperfusion (diagonally striped bars), and survival (lightly shadedbars) are shown. Means±SEM are indicated, with the numbers of animalsfrom which the percentage survival was calculated indicated above thesurvival bars. *=p<0.05 vs control antibody.

FIGS. 35A-35B. Validation of quantitative spectrophotometricintracerebral hemorrhage assay, in the absence (FIG. 35A) or presence(FIG. 35B) of brain tissue. FIG. 35A. Standard curve in which knownconcentrations of hemoglobin were reduced to cyanomethemoglobin, afterwhich the OD at 550 nm was measured. N=5 determinations at each point,with means±SEM shown. The equation for the best-fit line and r value areshown. FIG. 35B. Known concentrations of hemoglobin (using autologousblood diluted in saline) were added to fixed volumes of fresh braintissue homogenate and the spectrophotometric hemoglobin assay wasperformed. Brains were divided into hemispheres; for each animal, onehemisphere was immersed in physiological saline for 20 minutes (NS,solid line), and the other hemisphere was placed in triphenyltetrazoliumchloride (TTC, dashed line) for 20 minutes (similar to the procedurethat would be done to measure cerebral infarction volume). For eachconcentration of added hemoglobin, spectrophotometric hemoglobin assaywas performed on 6 hemispheres. Means±SEM are shown.

FIGS. 36A-36B. Quantitative spectrophotometric hemoglobin assay. FIG.36A. Effects of collagenase-infusion and rt-PA on murine quantitativeICH. Mice were stereotactically infused with ICH-inducing agents intothe right deep cortex/basal ganglia. Brains were harvested 24 h laterand the spectrophotometric hemoglobin assay was performed to quantifyICH. Mice were subjected to 1) no treatment (Control) 2), stereotacticinfusion of 1 μl normal saline solution (Sham), 3) stereotactic infusionof 0.024 μg collagenase B in 1 μl normal saline solution (Collagenase),or 4) stereotactic infusion of collagenase B (as above) followed byintravenous tissue plasminogen activator (1 mg/kg in 0.2 μl normalsaline solution) by dorsal penile vein injection (Collagenase+rt-PA).**p<0.001 vs. Sham or Control. FIG. 36B. Effect of rt-PA following focalischemic stroke on murine quantitative ICH. Mice were subjected to 45minutes of MCA occlusion followed by reperfusion and then 1) intravenous0.2 μl of normal saline solution (Stroke+Saline) or 2) intravenoustissue plasminogen activator (15 mg/kg in 0.2 μl normal saline solution)(Stroke+rt-PA). Brains were harvested 24 h later and thespectrophotometric hemoglobin assay was performed to quantify ICH.**p<0.05.

FIG. 37. Demonstration of the scoring system used for the visualdetermination of ICH following stroke. Each slice, taken from differentanimals subjected to stroke, represents the coronal slice of brain whichexhibits the maximal hemorrhagic diameter. The numbers correspond to thevisually determined hemorrhage score, as defined in the Methods section.

FIGS. 38A-38B. Visual ICH score. FIG. 38A. Effects ofcollagenase-infusion and rt-PA on murine visual. ICH score. Mice werestereotactically infused with ICH-inducing agents into the right deepcortex/basal ganglia. Mice were subjected to 1) no treatment (Control),2) stereotactic infusion of 1 μl normal saline solution (Sham), 3)stereotactic infusion of 0.024 μg collagenase B in 1 μl normal salinesolution (Collagenase), or 4) stereotactic infusion of collagenase B (asabove) followed by intravenous tissue plasminogen activator (1 mg/kg in0.2 μl normal saline solution) by dorsal penile vein injection(Collagenase+rt-PA). Brains were harvested 24 h later, sectioned into 1mm coronal slices, and scored by a blinded observer as described in theMethods section. *p<0.05 vs. Collagenase, p<0.005 vs. Sham or Control.FIG. 38B. Effect of rt-PA following focal ischemic stroke on murinevisual ICH score. Mice were subjected to 45 minutes of MCA occlusionfollowed by reperfusion and then 1) intravenous 0.2 μl of normal salinesolution (Stroke+Saline) or 2) intravenous tissue plasminogen activator(15 mg/kg in 0.2 μl normal saline solution) (Stroke+rt-PA). Brains wereharvested 24 h later, sectioned into 1 mm coronal slices, and scored bya blinded observer as described in the Methods section *p<0.01.Individual values for visual ICH scores are shown, with the median valuefor each group indicated by a horizontal line.

FIG. 39. Correlation between visual ICH and spectrophotometrichemoglobin assay. Optical density at 550 nm (ordinate) represents theresults obtained from the spectrophotometric hemoglobin assay in whichbrain tissue (from all experiments) was analyzed. The correspondingvisual ICH scores (as shown in FIG. 38) are plotted along the abscissa.For each point, mean±SEM are shown. Linear correlation was performedusing Pearson's linear correlation, with the equation of the line and rvalue shown.

FIGS. 40A-40F. FIG. 40A. Effect of stroke and Factor IXai administrationin stroke on the accumulation of radiolabeled platelets.¹¹¹Indium-platelets were administered either in control animals withoutstroke (n=4), or in animals immediately prior to stroke with (n=7) orwithout preoperative administration of Factor IXai (300 μg/kg, n=7).Platelet accumulation is expressed as the ipsilateral cpm/contralateralcpm. Means±SEM are shown. *p<0.05 vs No Stroke; **p<0.05 vsStroke+Vehicle. FIG. 40B. Accumulation of fibrin in infarcted cerebraltissue. Twenty-two hours following focal cerebral ischemia andreperfusion, a brain was harvested from a representative mouse which hadbeen pretreated prior to surgery with either vehicle (leftmost twolanes) or Factor IXai (300 μg/kg, rightmost two lanes). The brains weredivided into ipsilateral (R) and contralateral (L) hemispheres, andplasmin digestion performed to solubilize accumulated fibrin.Immunoblotting was performed using a primary antibody directed against aneoepitope expressed on the gamma—gamma chain dimer of crosslinkedfibrin. FIG. 40C-40F. Immunohistochemical identification of sites offibrin formation in stroke. Using the same antibody as described in FIG.2b to detect fibrin, brains were harvested from two mice followingstroke (upper and lower panels each represent a mouse). Arrows identifycerebral microvessels. Note that in both ipsilateral hemispheres (rightpanels), intravascular fibrin can be clearly identified by the redstain, which is not seen in the contralateral (left panels), nonischemichemispheres.

FIGS. 41A-41C. FIG. 41A. Effect of Factor IXai on relative CBF in amurine stroke model, measured by laser doppler. CBF in FactorIXai-treated animals (300 μg/kg, n=48, dashed line) is significantlyhigher at 24 hours than vehicle-treated controls (n=62). Means±SEM areshown. *p<0.05. FIG. 41B. Effect of Factor IXai on infarct volumes in amurine stroke model, measured by TTC-staining of serial coronalsections. Animals were given vehicle (n=62) or Factor IXai (300 μg/kg,n=48). Means±SEM are shown. *p<0.05. FIG. 41C. Dose-response of FactorIXai in stroke. Factor IXai was administered immediately prior to theonset of stroke, and cerebral infarct volumes determined as described inFIG. 41B above. N=62, 48, 6, and 6, for Vehicle, 300 μg/kg, 600 μg/kg,and 1200 μg/kg doses respectively. Means±SEM are shown. *p<0.05 vsvehicle-treated animals.

FIGS. 42A-42B. Effect of Factor IXai on Intracerebral hemorrhage. FIG.42A. Spectrophotometric hemoglobin assay was performed as described inthe Methods section. O.D. at 550 nm is linearly related to brainhemoglobin content^(11,12). FIG. 42B. Visually-determined ICH score by ablinded observer, as described in the methods section. ICH scorecorrelates with spectrophotometrically determined brain hemoglobincontent^(11,12). Means±SEM are shown. *p<0.05 vs vehicle-treatedanimals.

FIG. 43. Effect of timing of Factor IXai administration on cerebralinfarct volumes when given after the onset of stroke. Mice weresubjected to focal cerebral ischemia and reperfusion as described in theMethods section. The preocclusion administration (leftmost 2 bars) datais that shown FIG. 42B. In additional experiments to determine theeffects of Factor IXai administered after stroke, immediately followingwithdrawal of the intraluminal occluding suture, vehicle (normal saline,n=13) or Factor IXai (300 μg/kg, n=7) was administered intravenously.Cerebral infarct volumes (based on TTC-stained serial sections obtainedat 22 hrs) were determined. Means±SEM are shown. *p<0.05, **p<0.05 vsvehicle-treated animals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a method for treating an ischemicdisorder in a subject which includes administering to the subject of apharmaceutically acceptable form of a selectin antagonist in asufficient amount over a sufficient time period to prevent white bloodcell accumulation so as to treat the ischemic disorder in the subject.The selectin antagonist may be a peptide nimetic, a nucleic acidmolecule, a ribozyme, a polypeptide, a small molecule, a carbohydratemolecule, a monosaccharide, an oligosaccharide or an antibody. Theselectin may be a P-selectin, an E-selectin, or an L-selectin. Theantibody may be a P-selectin antibody. The antibody may further includea polyclonal antibody or a monoclonal antibody. The P-selectinantagonist may include a nitric oxide (NO) precursor such as L-arginine,an NO donor such as nitroglycerin or nitroprusside, a cyclic nucleotideanalog such as a cyclic GMP or cyclic AMP analog, or a phosphodiesteraseinhibitor.

The pharmaceutically acceptable form of P-selectin antagonist mayinclude a P-selectin antagonist and a pharmaceutically acceptablecarrier. The carrier may include an aerosol, intravenous, oral ortopical carrier.

The white blood cell may be a neutrophil or a monocyte. The subject maybe a mammal. The mammal may be a human, a cow, a pig, a sheep, a dog, acat, a monkey, a fowl or any animal model of a human disease ordisorder.

The ischemic disorder may include, but is not limited to a peripheralvascular disorder, a venous thrombosis, a pulmonary embolus, amyocardial infarction, a transient ischemic attack, unstable angina, areversible ischemic neurological deficit, sickle cell anemia or a strokedisorder.

The subject may be undergoing heart surgery, lung surgery, spinalsurgery, brain surgery, vascular surgery, abdominal surgery, or organtransplantation surgery. The organ transplantation surgery may includeheart, lung, pancreas or liver transplantation surgery.

The present invention further provides for a method for treating anischemic disorder in a subject which comprises administering to thesubject carbon monoxide gas in a sufficient amount over a sufficientperiod of time thereby treating the ischemic disorder in the subject.

The administration of carbon monoxide may be via inhalation by thesubject or via extracorporeal exposure to blood or body fluids of thesubject.

The amount of carbon monoxide which may be sufficient to treat thesubject includes but is not limited to from about 0.0001% carbonmonoxide in an inert gas to about 2% carbon monoxide in an inert gas.The inert gas may be oxygen, nitrogen, argon or air. In one embodimentof the present invention, the amount of carbon monoxide administered maybe 0.1% carbon monoxide in air.

The period of time sufficient to administer carbon monoxide to a subjectto treat an ischemic disorder includes but is not limited to from about1 day before surgery to about 1 day after surgery. The period of timemay be from about 12 hours before surgery to about 12 hours aftersurgery. The period of time may further include from about 12 hoursbefore surgery to about 1 hour after surgery. The period of time mayfurther include from about 1 hour before surgery to about 1 hour aftersurgery. The period of time may further include from about 20 minutesbefore surgery to about 1 hour after surgery. The period of timesufficient to treat an ischemic disorder in a subject who is notundergoing surgery may include before and during any physicalmanifestation of such disorder. Administration of carbon monoxide ispreferable before the manifestation in order to lessen suchmanifestation of an ischemic disorder. Carbon monoxide administrationhas been shown as described hereinbelow to be protective of ischemia ina subject if administered prior to surgery.

As used herein, the “ischemic disorder” encompasses and is not limitedto a peripheral vascular disorder, a venous thrombosis, a pulmonaryembolus, a myocardial infarction, a transient ischemic attack, lungischemia, unstable angina, a reversible ischemic neurological deficit,adjunct thromolytic activity, excessive clotting conditions, sickle cellanemia or a stroke disorder.

The subject may be undergoing heart surgery, lung surgery, spinalsurgery, brain surgery, vascular surgery, abdominal surgery, or organtransplantation surgery. The organ transplantation surgery may includeheart, lung, pancreas or liver transplantation surgery.

The carbon monoxide may be administered in an indirect manner. Ratherthan the subject directly inhaling or receiving carbon monoxide gas or agas mixture, the subject may be given compounds to stimulate the in vivoproduction of carbon monoxide. Such compounds may include but are notlimited to heme, ferritin, hematin, endogenous precursors to hemeoxygenase or heme oxygenase stimulators. In addition, the subject may beexposed to an environment of low oxygen level compared to the normalatmosphere.

Heme oxygenase is an endogenous enzyme which synthesizes carbon monoxidefrom precursor heme (it is part of the normal way in which heme isdegraded and metabolized in the body). When mice were exposed to eitherhypoxia or tissue ischemia, levels of both the messenger RNA which codesfor heme oxygenase protein and the protein itself were increased. Inaddition, the activity of the enzyme was increased, as indicated bymeasurements of carbon monoxide in the tissue.

Another embodiment of the present invention is a method for treating anischemic disorder in a subject which comprises administering to thesubject a pharmaceutically acceptable form of inactivated Factor IX in asufficient amount over a sufficient period of time to inhibitcoagulation so as to treat the ischemic disorder in the subject. Thesufficient amount may include but is not limited to from about 75 μg/kgto about 550 μg/kg. The amount may be 300 μg/kg. The pharmaceuticallyacceptable form of inactivated Factor IX includes inactivated Factor IXand a pharmaceutically acceptable carrier.

The Factor IX may be inactivated by the standard methods known to one ofskill in the art, such as heat inactivation. Factor IX may beinactivated or Factor IX activity may be inhibited by an antagonist.Such antagonist may be a peptide mimetic, a nucleic acid molecule, aribozyme, a polypeptide, a small molecule, a carbohydrate molecule, amonosaccharide, an oligosaccharide or an antibody.

The present invention provides for a method for identifying a compoundthat is capable of improving an ischemic disorder in a subject whichincludes: a) administering the compound to an animal, which animal is astroke animal model; b) measuring stroke outcome in the animal, and c)comparing the stroke outcome in step (b) with that of the stroke animalmodel in the absence of the compound so as to identify a compoundcapable of improving an ischemic disorder in a subject. The strokeanimal model includes a murine model of focal cerebral ischemia andreperfusion. The stroke outcome may be measured by physical examination,magnetic resonance imaging, laser doppler flowmetry, triphenyltetrazolium chloride staining, chemical assessment of neurologicaldeficit, computed tomography scan, or cerebral cortical blood flow. Thestroke outcome in a human may be measured also by clinical measurements,quality of life scores and neuropsychometric testing. The compound mayinclude a P-selectin antagonist, an E-selectin antagonist or anL-selectin antagonist.

The present invention further provides a method for identifying acompound that is capable of preventing the accumulation of white bloodcells in a subject which includes:a) administering the compound to ananimal, which animal is a stroke animal model; b) measuring strokeoutcome in the animal, and c) comparing the stroke outcome in step (b)with that of the stroke animal model in the absence of the compound soas to identify a compound capable of preventing the accumulation ofwhite blood cells in the subject.

The white blood cell may be, but is not limited to, a neutrophil, aplatlet or a monocyte. The compound may be but is not limited to aselectin inhibitor, a monocyte inhibitor, a platelet inhibitor or aneutrophil inhibitor. The selectin inhibitor may be but is not limitedto a P-selectin, E-selectin or L-selectin inhibitor. Selectins areexpressed on the surface of the platlets and such selectin inhibitors orantagonists as described herein may prevent the expression of suchselectins on the surface of the cell. The prevention of expression maybe at the transcriptional, translational, post-translational levels orpreventing the movement of such selectins through the cytosol andpreventing delivery at the cell surface.

The present invention provides for treatment of ischemic disorders byinhibiting the ability of the neutrophil, monocyte or other white bloodcell to adhere properly. This may be accomplished removing the counterligand, such as CD18. It has been demonstrated as discussed hereinbelow,that “knock-out” CD18 mice (mice that do not have expression of thenormal CD18 gene) are protected from adverse ischemic conditions. Theendothelial cells on the surface of the vessels in the subject may alsobe a target for treatment. In a mouse model of stroke, administration ofTPA as a thrombolytic agent caused some visible hemorrhaging along withimprovement of the stroke disorder. However, administration of aP-selectin antagonist also improved stroke disorder in the animal model,but without the coincident hemorrhaging. The present invention may beused in conjunction with a thrombolytic therapy to increase efficacy ofsuch therapy or to enable lower doses of such therapy to be administeredto the subject so as to reduce side effects of the thrombolytic therapy.

As used herein, the term “suitable pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers, suchas phosphate buffered saline solution, water, emulsions such as anoil/water emulsion or a triglyceride emulsion, various types of wettingagents, tablets, coated tablets and capsules. An example of anacceptable triglyceride emulsion useful in intravenous andintraperitoneal administration of the compounds is the triglycerideemulsion commercially known as Intralipid®.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients.

This invention also provides for pharmaceutical compositions includingtherapeutically effective amounts of protein compositions and compoundscapable of treating stroke disorder or improving stroke outcome in thesubject of the invention together with suitable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers useful in treatmentof neuronal degradation due to aging, a learning disability, or aneurological disorder. Such compositions are liquids or lyophilized orotherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),solubilizing agents (e.g., glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the compound, complexation withmetal ions, or incorporation of the compound into or onto particulatepreparations of polymeric compounds such as polylactic acid, polglycolicacid, hydrogels, etc, or onto liposomes, micro emulsions, micelles,unilamellar or multi lamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance of the compound or composition. The choice of compositionswill depend on the physical and chemical properties of the compoundcapable of alleviating the symptoms of the stroke disorder or improvingthe stroke outcome in the subject.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended bythe invention are particulate compositions coated with polymers (e.g.,poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

Portions of the compound of the invention may be “labeled” byassociation with a detectable marker substance (e.g., radiolabeled with¹²⁵I or biotinylated) to provide reagents useful in detection andquantification of compound or its receptor bearing cells or itsderivatives in solid tissue and fluid samples such as blood, cerebralspinal fluid or urine.

When administered, compounds are often cleared rapidly from thecirculation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of bioactive compounds may be required to sustaintherapeutic efficacy. Compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline areknown to exhibit substantially longer half-lives in blood followingintravenous injection than do the corresponding unmodified compounds(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987).Such modifications may also increase the compound's solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound adducts less frequently or in lower doses thanwith the unmodified compound.

Attachment of polyethylene glycol (PEG) to compounds is particularlyuseful because PEG has very low toxicity in mammals (Carpenter et al.,1971). For example, a PEG adduct of adenosine deaminase was approved inthe United States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. A second advantage afforded by theconjugation of PEG is that of effectively reducing the immunogenicityand antigenicity of heterologous compounds. For example, a PEG adduct ofa human protein might be useful for the treatment of disease in othermammalian species without the risk of triggering a severe immuneresponse. The compound of the present invention capable of alleviatingsymptoms of a cognitive disorder of memory or learning may be deliveredin a microencapsulation device so as to reduce or prevent an host immuneresponse against the compound or against cells which may produce thecompound. The compound of the present invention may also be deliveredmicroencapsulated in a membrane, such as a liposome.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl groupsof the carboxy-terminal amino acid, tyrosine side chains, or toactivated derivatives of glycosyl chains attached to certain asparagine,serine or threonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

By means of well-known technique such as titration and by taking intoaccount the observed pharmacokinetic characteristics of the agent in theindividual subject, one of skill in the art can determine an appropriatedosing regimen. See, for example, Benet, et al., “ClinicalPharmacokinetics” in ch. 1 (pp. 20-32) of Goodman and Gilman's ThePharmacological Basis of Therapeutics, 8th edition, A. G. Gilman, et al.eds. (Pergamon, New York 1990).

The present invention provides for a pharmaceutical composition whichcomprises an agent capable of treating a stroke disorder or improvingstroke outcome and a pharmaceutically acceptable carrier. The carriermay include but is not limited to a diluent, an aerosol, a topicalcarrier, an aqueous solution, a nonaqueous solution or a solid carrier.

This invention is illustrated in the Experimental Detail section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS

Abbreviations: EC, endothelial cell; PMN, polymorphonuclear leukocyte;WP, Weibel-Palade body; vWF, von Willebrand factor; EGTA, ethyleneglycolbis (aminoethylether)tetraacetic acid; HBSS, Hank's balanced saltsolution; CS, coronary sinus; IL, interleukin; PAF, platelet activatingfactor; ICAM-1, intercellular adhesion molecule-1; HUVEC, humanumbilical vein EC; LR, lactated Ringer's solution; MCAO, middle cerebralartery occlusion; rt-PA, recombinant tissue plasminogen activator; HO-Ior HOI or HO I, heme oxygenase I; ICH, intracerebral hemorrhage; OD,optical density; MCA, middle cerebral artery; rt-PA, recombinanttissue-type plasminogen activator; TIA, transient ischemic attack; TTC,triphenyltetrazolium chloride.

EXAMPLE 1 Cerebral Protection in Homozygous Null ICAM-1 Mice FollowingMiddle Cerebral Artery Occlusion: Role of Neutrophil Adhesion in thePathogenesis of Stroke

To investigate whether polymorphonuclear leukocytes (PMNs) contribute toadverse neurologic sequelae and mortality following stroke, and to studythe potential role of the leukocyte adhesion molecule IntercellularAdhesion Molecule-1 (ICAM-1) in the pathogenesis of stroke, a murinemodel of transient focal cerebral ischemia was employed consisting ofintraluminal middle cerebral artery (MCA) occlusion for 45 minutesfollowed by 22 hours of reperfusion. PMN accumulation, monitored bydeposition of ¹¹¹Indium-labelled PMNs in postischemic cerebral tissue,was increased 2.5 fold in the ipsilateral (infarcted) hemispherecompared with the contralateral (noninfarcted) hemisphere (p<0.01). Miceimmunodepleted of neutrophils prior to surgery demonstrated a 3.0-foldreduction in infarct volumes (p<0.001), based on triphenyltetrazoliumchloride staining of serial cerebral sections, improved ipsilateralcortical cerebral blood flow (measured by laser doppler) and reducedneurological deficit compared with controls. In wild type mice subjectedto 45 minutes of ischemia followed by 22 hours of reperfusion, ICAM-1mRNA was increased in the ipsilateral hemisphere, withimmunohistochemistry localizing increased ICAM-1 expression on cerebralmicrovascular endothelium. The role of ICAM-1 expression in stroke wasinvestigated in homozygous null ICAM-1 mice (ICAM-1 −/−) in comparisonwith wild type controls (ICAM-1 +/+). ICAM-1 −/− mice demonstrated a3.7-fold reduction in infarct volume (p<0.005), a 35% increase insurvival (p<0.05), and reduced neurologic deficit compared with ICAM-1+/+ controls. Cerebral blood flow to the infarcted hemisphere was3.1-fold greater in ICAM-1 −/− mice compared with ICAM-1 +/+ controls(p<0.01), suggesting an important role for ICAM-1 in the genesis ofpost-ischemic cerebral no-reflow. Because PMN-depleted and ICAM-1deficient mice are relatively resistant to cerebral ischemia-reperfusioninjury, these studies suggest an important role for ICAM-1 mediated PMNadhesion in the pathophysiology of evolving stoke.

Introduction

Neutrophils (PMNs) are critically involved in the earliest stages ofinflammation following tissue ischemia, initiating scavenger functionswhich are later subsumed by macrophages. There is a darker side toneutrophil influx, however, especially in postischemic tissues¹⁻⁷, whereactivated PMNs may augment damage to vascular and parenchymal cellularelements. Experimental evidence points to a pivotal role for endothelialcells in establishing postischemic PMN recruitment, in thathypoxic/ischemic endothelial cells synthesize the proinflammatorycytokine IL-1⁸ as well as the potent neutrophil chemoattractant andactivator IL-8⁹. Firm adhesion of PMNs to activated endothelium in apostischemic vascular milieu is promoted by translocation of P-selectinto the cell surface¹⁰ as well as enhanced production of plateletactivating factor and ICAM-1¹¹.

While strategies to block each of these mechanisms of neutrophilrecruitment are protective in various models of ischemia and reperfusioninjury, their effectiveness in cerebral ischemia/reperfusion injuryremains controversial. There is considerable evidence that in the brain,as in other tissues, an early PMN influx follows an ischemicepisode¹²⁻¹⁷. Immunohistochemical studies have described increasedexpression of the PMN adhesion molecules P-selectin and intracellularadhesion molecule-1 (ICAM-1) in the postischemic cerebralvasculature^(12,18-20). The pathogenic relevance of adhesion moleculeexpression in the brain remains controversial, however; data from atrial of monoclonal anti-ICAM-1 antibody in stroke in humans is not yetavailable. In animal models, there is conflicting experimental evidenceregarding the effectiveness of anti-adhesion molecule strategies in thetreatment of experimental stroke²¹⁻²³. To determine whether ICAM-1participates in the pathogenesis of postischemic cerebral injury, theexperiments reported here were undertaken in a murine model of focalcerebral ischemia and reperfusion so that the role of a single, criticalmediator of PMN adhesion (ICAM-1) could be determined. These studiesdemonstrate that enhanced ICAM-1 expression and neutrophil influx followan episode of focal cerebral ischemia. Furthermore, these studies showthat both neutrophil-deficient and transgenic ICAM-1 null mice arerelatively resistant to cerebral infarction following ischemia andreperfusion, providing strong evidence for an exacerbating role ofICAM-1 in the pathophysiology of stroke.

Materials and Methods

Mice: Experiments were performed with transgenic ICAM-1 deficient micecreated as previously reported²⁴ by gene targeting in J1 embryonic stemcells, injected into C57BL/6 blastocysts to obtain germlinetransmission, and backcrossed to obtain homozygous null ICAM-1 mice. Allexperiments were performed with ICAM-1 −/− or wild-type (ICAM-1+/+)cousin mice from the fifth generation of backcrossings with C57BL/6mice. Animals were seven to nine weeks of age and weighed between 25-36grams at the time of experiments. For certain experiments, neutrophildepletion of C57BL/6 mice was accomplished by administering polyclonalrabbit anti-mouse neutrophil antibody²⁵ (Accurate Scientific, Westbury,N.Y.) preadsorbed to red blood cells as a daily intraperitonealinjection (0.3 mL of 1:12 solution) for 3 days. Experiments in thesemice were performed on the fourth day after confirming agranulocytosisby Wright-Giemsa stained peripheral blood smears.

Transient Middle Cerebral Artery Occlusion²⁶: Mice were anesthetizedwith an intraperitoneal injection of 0.3 ml of ketamine (10 mg/cc) andxylazine (0.5 mg/cc). Animals were positioned supine on arectal-temperature controlled operating surface (Yellow SpringsInstruments, Inc., Yellow Springs, Ohio). Animal core temperature wasmaintained at 36-38° C. intraoperatively and for 90 minutespost-operatively. Middle cerebral artery occlusion was performed asfollows; A midline neck incision was created to expose the right carotidsheath under the operating microscope (16-25× zoom, Zeiss, Thornwood,N.Y.). The common carotid artery was freed from its sheath, and isolatedwith a 4-0 silk, and the occipital and pterygopalatine arteries wereeach isolated and divided. Distal control of the internal carotid arterywas obtained and the external carotid was cauterized and divided justproximal to its bifurcation into the lingual and maxillary divisions.Transient carotid occlusion was accomplished by advancing a 13 mmheat-blunted 5-0 nylon suture via the external carotid stump to theorigin of the middle cerebral artery. After placement of the occludingsuture, the external carotid artery stump was cauterized to preventbleeding through the arteriotomy, and arterial flow was reestablished.In all cases, the duration of carotid occlusion was less than twominutes. After 45 minutes, the occluding suture was withdrawn toestablish reperfusion. These procedures have been previously describedin detail²⁶.

Measurement of cerebral cortical blood flow. Transcranial measurementsof cerebral blood flow were made using laser doppler flow measurements(Perimed, Inc., Piscataway, N.J.) after reflection of the skin overlyingthe calvarium, as previously described²⁷ (transcranial readings wereconsistently the same as those made after craniectomy in pilot studies).Using a 0.7 mm straight laser doppler probe (model #PF303, Perimed,Piscataway, N.J.) and previously published landmarks (2 mm posterior tothe bregma, 6 mm to each side of midline), relative cerebral blood flowmeasurements were made as indicated; immediately after anesthesia, afterocclusion of the middle cerebral artery, immediately after reperfusion,and at 24 hours just prior to euthanasia. Data are expressed as theratio of the doppler signal intensity of the ischemic compared with thenonischemic hemisphere. Although this method does not quantify cerebralblood flow per gram of tissue, use of laser doppler flow measurements atprecisely defined anatomic landmarks serves as a means of comparingcerebral blood flows in the same animal serially over time. The surgicalprocedure/intraluminal middle cerebral artery occlusion and reperfusionwere considered to be technically adequate if ≦50% reduction in relativecerebral blood flow was observed immediately following placement of theintraluminal occluding suture and a ≦33% increase in flow over baselineocclusion was observed immediately following removal of the occludingsuture. These methods have been used in previous studies²⁶.

Preparation and administration of ¹¹¹In-labelled of murine neutrophils:

Citrated blood from wild type mice was diluted 1:1 with NaCl (0.9%)followed by gradient ultracentrifugation on Ficoll-Hypaque (Pharmacia,Piscataway, N.J.). Following hypotonic lysis of residual erythrocytes(20 sec exposure to distilled H₂O followed by reconsitution with 1.8%NaCl), the neutrophils were suspended in phosphate buffered saline(PBS). 5-7.5×10⁶ neutrophils were suspended in PBS with 100 μCi of¹¹¹Indium oxine (Amerhsam Mediphysics, Port Washington, N.Y.) for 15minutes at 37° C. After washing with PBS, the neutrophils were gentlypelleted (450 g), and resuspended in PBS to a final concentration of1.0×10⁶ cells/ml. Immediately prior to surgery, 100 μL of radiolabelledPMNs admixed with physiologic saline to a total volume of 0.3 mL (≈3×10⁶cpm) was administered by penile vein injection. Following humaneeuthanasia, brains were obtained as described, and neutrophil depositionquantified as cpm/gm of each hemisphere.

Neurological Exam:

Twenty-four hours after middle cerebral artery occlusion andreperfusion, prior to giving anesthesia, mice were examined forneurological deficit using a four-tiered grading system²⁶: A score of(1) was given if the animal demonstrated normal spontaneous movements; ascore of (2) was given if the animal was noted to be turning to theright (clockwise circles) when viewed from above (i.e., towards thecontralateral side); a score of (3) was given if the animal was observedto spin longitudinally (clockwise when viewed from the tail); a score of(4) was given if the animal was crouched on all fours, unresponsive tonoxious stimuli. This scoring system has been previously described inmice²⁶, and is based upon similar scoring systems used in rats^(28,29)which are based upon the contralateral movement of animals with stroke;following cerebral infarction, the contralateral side is “weak” and sothe animal tends to turn towards the weakened side. Previous work inrats²⁸ and mice²⁶ demonstrates that larger cerebral infarcts areassociated with a greater degree of contralateral movement, up to thepoint where the infarcts are so large that the animal remainsunresponsive.

Calculation of Infarct Volume

After neurologic examination, mice were given 0.3 mL of ketamine (10mg/ml) and xylazine (0.5 mg/ml), and final cerebral blood flowmeasurements were obtained. Humane euthanasia was performed bydecapitation under anesthesia, and brains were removed and placed in amouse brain matrix (Activational Systems Inc., Warren, Mich.) for 1 mmsectioning. Sections were immersed in 2% 2,3,5, -triphenyl,2H-tetrazolium chloride (TTC, Sigma Chemical Co., St. Louis, Mo.) in0.9% phosphate-buffered saline, incubated for 30 minutes at 37° C., andplaced in 10% formalin^(26,30-32). Infarcted brain was visualized as anarea of unstained tissue, in contrast to viable tissue, which stainsbrick red. Infarct volumes were calculated from planimetered serialsections and expressed as the percentage of infarct in the ipsilateralhemisphere.

RNA extraction and Northern blot analysis

24 hours following focal ischemia and reperfusion, brains were obtainedand divided into ipsilateral (infarct) and contralateral (noninfarct)hemispheres. To detect ICAM-1 transcripts, total RNA was extracted fromeach hemisphere using an RNA isolation kit (Stratagene, La Jolla,Calif.). Equal amounts of RNA (20 μg/lane) were loaded onto a 1.4%agarose gel containing 2.2 M formaldehyde for size fractionation, andthen transferred overnight to nylon (Nytran) membranes with 10× SSCbuffer by capillary pressure. A murine ICAM-1 cDNA probe³³ (1.90 kb,ATCC, Rockville, Md.) was labelled with ³²P-α-dCTP by random primerlabelling (Prime-A-Gene kit, Promega), hybridized to blots at 42° C.,followed by 3 washes of 1× SSC/0.05% SDS. Blots were developed withinX-Omat AR film exposed with light screens at −70° C. for 7 days. Aβ-actin probe (ATCC) was used to confirm equal RNA loading.

Immunohistochemistry

Brains were removed at the indicated times following middle cerebralartery occlusion, fixed in 10% formalin, paraffin embedded and sectionedfor immunohistochemistry. Sections were stained with a rat anti-murineICAM-1 antibody (1:50 dilution, Genzyme, Cambridge, Mass.), and sites ofprimary antibody binding were visualized by an alkaline phosphataseconjugated secondary antibody detected with FastRed (TR/naphthol AS-MX,Sigma Chemical Co., St. Louis, Mo.).

Data Analysis

Cerebral blood flow, infarct volumes and neurologic outcome scores werecompared using Student's t-test for unpaired variables.¹¹¹Indium-neutrophil deposition was evaluated as paired data [comparingcontralateral (noninfarct) to ipsilateral (infarct) hemisphere], tocontrol for variations in injected counts or volume of distribution.Survival differences between groups was tested using contingencyanalysis with the Chi-square statistic. Values are expressed asmeans±SEM, with a p<0.05 considered statistically significant.

Results:

Neutrophil Accumulation in Stroke

Previous pathologic studies have shown neutrophil accumulation followingcerebral infarction^(15-17,34-36). To determine whether neutrophilsaccumulate in the murine model of focal cerebral ischemia andreperfusion, neutrophil accumulation following transient (45 min)ischemia and reperfusion (22 hrs) was quantified by measuring thedeposition of ¹¹¹In-labeled neutrophils given to wild-type mice prior tothe ischemic event. These experiments demonstrated significantly greaterneutrophil accumulation (2.5-fold increase) in the ipsilateral(infarcted) compared with the contralateral (noninfarcted) hemispheres(in=7, p<0.01; FIG. 1). Similar results were obtained when neutrophilinflux was monitored by myeloperoxidase assays, though low levels ofactivity were recorded in the latter assay (data not shown).

Effect of Neutrophil Depletion on Stroke Outcome

To determine the effect of neutrophil influx on indices of strokeoutcome, mice were immunodepleted of neutrophils beginning three daysprior to surgery. When surgery was performed on the fourth day, nearlycomplete agranulocytosis was evident on smears of peripheral blood.Neutropenic mice (n=18) were subjected to 45 min cerebral ischemia and22 hours of reperfusion, and indices of stroke outcome determined.Infarct volumes were 3-fold smaller in neutropenic animals compared withwild type controls (11.1±1.6% vs 33.3±6.4%, p<0.001; FIG. 2A). Thedecrease in infarct volumes in neutropenic mice was paralleled byreduced neurologic deficit scores (FIG. 2B), increased post-reperfusioncerebral cortical blood flows (FIG. 2C), and a trend towards reducedovernight mortality (22% mortality in neutropenic mice vs 50% mortalityin controls, FIG. 2D).

ICAM-1 Expression in Murine Stroke

To establish the effect of cerebral ischemia/reperfusion in the murinemodel, ICAM-1 mRNA levels were evaluated following cerebral ischemia andreperfusion in wild type mice. Ipsilateral (infarcted) cerebralhemisphere demonstrated increased ICAM-1 mRNA by Northern blot analysiscompared with RNA obtained from the contralateral (noninfarcted)hemisphere from the same animal (FIG. 3). To evaluate ICAM-1 antigenexpression in this murine model, wild type mice were subjected to 45minutes of ischemia followed by 23 hours of reperfusion, and thecerebral microvasculature examined by immunohistochemistry. ICAM-1antigen expression was not detectable in the cerebral microvasculaturecontralateral to the infarct (FIG. 4A), but was markedly increased onthe ipsilateral side, with prominent ICAM-1 staining of cerebralendothelial cells (FIG. 4B).

Role of ICAM-1 in Stroke

To explore the role of ICAM-1 in stroke, transgenic mice which werehomozygous ICAM-1 deficient²⁴ were studied in the murine model of focalcerebral ischemia and reperfusion. Because variations in cerebrovascularanatomy have been reported to result in differences in susceptibility toexperimental stroke in mice³⁷, India ink staining was performed on theCircle of Willis in homozygous null (ICAM-1 −/−) and ICAM-1 +/+ mice.These experiments (FIG. 5) demonstrated that there were no grossanatomic differences in the vascular pattern of the cerebralcirculation. To determine the role of the intercellular adhesionmolecule-1 in neutrophil influx following focal cerebral ischemia andreperfusion, neutrophil accumulation was measured in homozygous nullICAM-1 mice (ICAM-1 −/−) mice (n=14) and wild-type controls (n=7)infused with ¹¹¹In-labeled neutrophils. Relative neutrophil accumulation(ipsilateral cpm/contralateral cpm) was diminished (39% reduction) inthe ICAM-1 −/− mice compared with ICAM-1 +/+ controls (1.70±0.26 vs.2.9±0.52, p<0.05).

Experiments were the performed to investigate whether expression ofICAM-1 has a pathophysiologic role in outcome following stroke. ICAM-1−/− mice (n=13) were significantly protected from the effects of focalcerebral ischemia and reperfusion, based on a 3.7-fold reduction ininfarct volume (p<0.01) compared with ICAM-1 +/+ controls (FIGS. 6 and7A). This reduction in infarct volume was accompanied by reducedneurologic deficit (FIG. 7B) and increased post-reperfusion cerebralcortical blood flow (FIG. 7C). Given these results, it was notsurprising that mortality was also significantly decreased in the theICAM-1 −/− mice compared with ICAM-1 +/+ controls (15% vs. 50%, p<0.05;FIG. 7D).

Discussion

Epidemiologic evidence in humans suggests that neutrophils contribute toboth the initiation of stroke³⁸ as well as to cerebral tissue injury andpoor clinical outcome³⁹, with a potential role for neutrophils inpostischemic hypoperfusion, neuronal dysfunctions, and scarformation⁴⁰⁻⁴⁴. Although there is considerable experimental evidencewhich suggests that neutrophils can exacerbate tissue damage followingstroke^(13,45-48), certain pieces of experimental data have stokedcontroversy by failing to find an association between agents which blockneutrophil accumulation and indices of stroke outcome. In a rat model ofstroke, antibody-mediated depletion of neutrophils prior to strokesignificantly decreased brain water content and infarct size¹³. However,cyclophosphamide-induced leukocytopenia in a gerbil model⁴⁹ oranti-neutrophil antibody administration to dogs⁵⁰ showed no beneficialeffects in global models of cerebral ischemia. Experimental therapytargeted at interfering with neutrophil-endothelial interactions hasalso produced mixed results. In a feline model of transient focalcerebral ischemia, treatment with antibody to CD18 (the common subunitof β₂ integrins, which bind to intercellular adhesion molecule-1⁵¹) didnot alter recovery of cerebral blood flow, return of evoked potentials,or infarct volume²³. Other experiments, however, have found thatmicrovascular patency after transient focal ischemia in primates isimproved by antibodies to CD18¹⁴. In a similar rat model,anti-CD11b/CD18 antibody has also been shown to reduce both neutrophilaccumulation and ischemia-related neuronal damage⁵².

The experiments reported here show that in a murine model of focalcerebral ischemia and reperfusion, neutrophils accumulate inpostischemic cerebral tissue, a finding corroborated in other modelswhich similarly demonstrate increased granulocyte accumulation in areasof low cerebral blood flow early during the post-ischemicperiod^(15,16,36,45). Not only do neutrophils accumulate during thepost-ischemic period in mice, but their presence exacerbates indices ofstroke outcome. When animals were made neutropenic prior to the ischemicevent, cerebral infarcts were smaller, with improved cerebral perfusionfollowing the ischemic event. These data are quite similar to thatreported in a rabbit model of thromboembolic stroke, in whichimmunodepletion of neutrophils resulted both in reduced infarctionvolume and improved blood flow³⁵. Because neutrophils contribute tomurine post-ischemic cerebral injury, a strategy was pursued toelucidate the role of ICAM-1 in the pathophysiology of stroke usingdeletionally mutant ICAM-1 mice²⁴. Experiments indicate that homozygousnull ICAM-1 mice are relatively resistant to the deleterious effects ofcerebral ischemia and reperfusion.

To demonstrate the role of both neutrophils and ICAM-1 in thepathogenesis of tissue injury in stroke, the studies reported here usedseveral methods for assessing stroke outcome. Although numerousinvestigators have used TTC staining to quantify cerebral infarctvolumes^(36,30-32,37,53), there has been some controversy as to theaccuracy of this method, especially when evaluated early following theischemic event. In the TTC method, 2,3,5 triphenyl, 2H-tetrazoliumchloride (TTC) reacts with intact oxidative enzymes on mitochondrialcristae and is thereby reduced to a colored formazan⁵⁴. TTC staining isunreliable before 2 hours of ischemia have elapsed; beyond 36 hours,cells infiltrating into the infarcted tissue can stain positively withTTC, thereby obscuring the clear demarcation between infarcted andnoninfarcted tissues seen with earlier staining³¹. Although the size ofthe infarct delineated by TTC staining correlates well with infarct sizedelineated by hematoxylin and eosin staining^(30,32), directmorphometric measurements tend to overestimate infarct volumes due tocerebral edema, especially during the first 3 days following theischemic event³². Even given these limitations, however, the studiesreported here incorporate three additional methods to define the role ofneutrophils and ICAM-1 in stroke outcome, including neurologic deficitscore, relative cerebral blood flow to the affected area, and mortality.These additional measures, which do not depend upon the accuracy of TTCstaining, contribute strongly to the identification of a pathogenic rolefor both neutrophils and ICAM-1 in stroke.

There has been a recent profusion of scientific studies exploring themechanistic basis for neutrophil recruitment to post-ischemic tissues.Endothelial cells appear to be the chief regulators for neutrophiltraffic, regulating the processes of neutrophil chemoattraction,adhesive, and emigration from the vasculature⁵⁵. When exposed to ahypoxic environment as a paradigm for tissue ischemia, endothelial cellssynthesize the potent neutrophil chemoattractant and activatorInterleukin-8 (IL-8)⁹, the blockade of which appears to be beneficial ina lung model of ischemia and reperfusion⁶. In addition, hypoxicendothelial cells synthesize the proinflammatory cytokineInterleukin-1⁸, which can upregulate endothelial expression of theneutrophil adhesion molecules E-selectin and ICAM-1 in an autocrinefashion^(8,9,56). Other neutrophil adhesion mechanisms may also beactivated in the brain following ischemia, such as release of P-selectinfrom preformed storage pools within Weibel-Palade body membranes¹⁰. In aprimate model, P-selectin expression was rapidly and persistentlyenhanced following focal middle cerebral artery ischemia andreperfusion¹⁸. Although P-selectin-dependent neutrophil recruitmentappears to be deleterious following cardiac ischemia and reperfusion⁵⁷,its pathophysiologic relevance in the setting of stroke has not yet beendetermined. While hypoxia induces de novo synthesis of the bioactivelipid platelet activating factor (PAF)¹¹, in a spinal cord ischemiareperfusion model, PAF antagonism offered no incremental benefit whengiven simultaneously with antibody to CD11/CD18⁴⁸.

Understanding the role of ICAM-1 in the pathophysiology of strokeappears to be of particular relevance in humans for several reasons.Increased cerebrovascular ICAM-1 expression has been demonstrated inprimates by 4 hours of ischemia and reperfusion, particularly in thelenticulostriate microvasculature¹⁸. An autopsy study of recent cerebralinfarcts in humans also demonstrated increased ICAM-1 expression²⁰. Asrats also express cerebral vascular ICAM-1 within 24 hours in both aphotochemically-induced model of rat cerebral ischemia¹⁹ as well as amiddle cerebral artery occlusion model¹², these data suggested thepotential usefulness of transgenic ICAM-1 deficient mice in elucidatingthe pathophysiolgic significance of increased post-cerebral ischemicICAM-1 expression. In particular, the time frame of ICAM-1 expression(increased by 4 to 24 hours in these models suggests that ICAM-1mediated neutrophil-endothelial interactions may be targeted in futurepharmacologic strategies to improve human stroke outcome, as this timeframe represents a realistic clinical window for therapeuticintervention.

Although neutropenic animals demonstrated increased regional cerebralblood flow compared with controls, compared with neutropenic animals,ICAM-1 deficient mice tended to have even higher ipsilateral cerebralblood flows at 24 hours. This observation may relate to the no-reflowphenomenon, wherein blood flow fails to return to pre-obstruction levelseven following release of a temporary vascular occlusion. A significantbody of previous work has implicated neutrophil plugging of capillarymicrovascular beds in this process⁵⁸, although in a model of globalcerebral ischemia, an 85% reduction in the circulating leukocyte countdid not decrease the incidence or severity of reflow failure⁴⁹. The datasuggest that non-neutrophil-dependent mechanisms, which neverthelessinvolve ICAM-1, may contribute to cerebrovascular post-ischemicno-reflow. As macrophages and lymphocytes both express LFA-1, whichmediates an adhesive interaction with endothelial cell ICAM-1⁵¹, it ispossible that ICAM-1 deficient mice have diminished recruitment of thesemononuclear cells, a possibility which is currently the subject offurther investigation. This hypothesis is supported by multiplepathologic observations demonstrating macrophage and lymphocyteaccumulation by 1-3 days following cerebral infarction^(12,17,19,34,59).

Taken together, the studies indicate that in a murine model of focalcerebral ischemia and reperfusion, neutrophils accumulate in theinfected hemisphere, and that neutropenic animals demonstrate cerebralprotection. Increased expression of ICAM-1 on cerebral endothelial cellsappears to be an important mechanism driving this neutrophilrecruitment, and mice which are unable to express ICAM-1 demonstrateimproved post-ischemic blood flows, reduced infarction volumes, andreduced mortality. These data suggest that pharmacologic strategiestargeted at interfering with neutrophil-endothelial interactions mayimprove the outcome following stroke in humans.

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EXAMPLE 2 Hypoxia-Induced Exocytosis of Endothelial Cell Weibel-PaladeBodies: A Mechanism for Rapid Neutrophil Recruitment Following CardiacPreservation

The period of hypoxia (H) is an important priming event for the vasculardysfunction which accompanies reperfusion, with endothelial cells (ECs)and neutrophils (PMNs) playing a central role. It was hypothesized thatEC Weibel-Palade (WP) body exocytosis during the hypoxic/ischemic periodduring organ preservation permits brisk PMN recruitment intopost-ischemic tissue, a process further amplified in an oxidant-richmileu. Exposure of human umbilical vein ECs to an hypoxic environment(pO₂≈20 torr) stimulated release of von Willebrand factor (vWF), storedin EC WP bodies, as well as increased expression of the WP body-derivedPMN-adhesion molecule P-selectin at the EC surface. Increased binding of¹¹¹In-labelled PMNs to hypoxic EC monolayers (compared with normoxiccontrols) was blocked with a blocking antibody to P-selectin, but wasnot effected by a nonblocking control antibody,. Although increasedP-selectin expression and vWF release were also noted duringreoxygenation, H alone (even in the presence of antioxidants) wassufficient to increase WP body exocystosis. To determine the relevanceof these observations to hypothermic cardiac preservation, during whichthe pO₂ within the cardiac vasculature declines to similarly low levels,experiments were performed in a rodent (rat and mouse) cardiacpreservation/transplantation model. Immunodepletion of recipient PMNs oradministration of a blocking anti-P-selectin antibody prior totransplantation resulted in reduced graft neutrophil infiltration andimproved graft survival, compared with identically preserved heartstransplanted into control recipients. To establish the important role ofendothelial P-selectin expression on the donor vasculature, murinecardiac transplants were performed using homozygous P-selectin deficientand wild type control donor hearts flushed free of blood/platelets priorto preservation/transplantation. P-selectin null hearts transplantedinto wild-type recipients demonstrated a marked (13-fold) reduction ingraft neutrophil infiltration and increased graft survival compared withwild type hearts transplanted into wild type recipients. To determinewhether coronary endothelial Weibel-Palade body exocytosis may occurduring cardiac preservation in humans, the release of vWF into thecoronary sinus was measured in 32 patients during open heart surgery.Coronary sinus samples obtained at the start and conclusion of theischemic period demonstrated an increase in coronary sinus vWF antigen(by ELISA) consisting of predominantly high molecular weight multimers(by immunoelectrophoresis). These suggest that EC Weibel-Palade bodyexocytosis occurs during hypothermic cardiac preservation, priming thevasculature of rapidly recruit PMNs during reperfusion.

Introduction

Endothelial cells (EC) adapt to hypoxia with a characteristic repertoireof responses (1), ranging from increased expression of endothelin (2) toincreased synthesis of basic fibroblast growth factor (3). Recentstudies have indicated that many features of the EC response to hypoxiaparallel features of the inflammatory response; hyposia selectivelyupregulates EC expression of Interleukins-1 (4), 6 (5), and 8 (6),platelet activating factor (PAF) (7,8), and ICAM-1 (4), which serve tofuel neutrophil (PMN) recruitment, adhesion, and activation at ischemicloci. Although these mechanism may explain the later phases ofreperfusion injury, the rapidity with which PMNs are recruited toreperfused myocardium following a period of hypothermic preservationsuggests that mechanism are involved which do not require de novoprotein synthesis. In this regard, P-selectin may figure prominently inthe earliest phases of PMN adhesion to the reperfused vasculature, asECs may rapidly express pre-formed P-selectin from subplasmalemmalstorage sites in Weibel-Palade body (9) membranes in response to theabundant oxygen free-radials generated in the reperfusion milieu(10-12). Furthermore, recent data have pointed to a role forP-selectin-mediated leukocyte arrest in leukostasis and tissue damageassociated with lung injury (12) and cardiac ischemia (14). Takentogether, these findings led to the hypothesis that the hypoxic/ischemicperiod associated with hypothermic myocardial preservation primes thevasculature of its characteristic response during reperfusion bydisplaying P-selectin prominently at the EC surface prior toreperfusion, serving as a spark which ignites and amplifies thesubsequent inflammatory response.

The experiments were designed to establish whether hypoxia per se (orhypothermic cardiac preservation, as occurs during cardiac surgery, inwhich the pO₂ within the coronary bed declines to pO₂<20 Torr) (15)would result in WP body exocytosis. Furthermore, experiments wereundertaken to determine the role of P-selectin-dependent PMN adhesion inthe cardiac graft failure which characteristically follows a period ofprolonged hypothermic preservation. The results shows that hypoxia issufficient to induce EC WP body exocytosis, even in the absence ofreoxygenation (and presence of antioxidants), and that the resultingP-selectin expression causes ECs to bind PMNs in vitro.

In rodents, the adverse consequences of P-selectin expression followinghypothermic cardiac preservation can be completely abrogated by eitherneutrophil depletion, P-selectin blockade, or by transplanting heartswhose endothelial cells fail to express P-selectin. Because WP bodyexocytosis also occurs in patients undergoing open heart surgery duringthe period of hypothermic cardiac preservation, these data suggest thatP-selectin blockade may represent a target for pharmacologicalintervention to improve cardiac preservation in humans.

Methods

Endothelial cell culture and exposure of cells to H or H/R

Human umbilical vein ECs were prepared from umbilical cords and grown inculture by the method of Jaffe (16) as modified by Thornton (17).Experiments utilized confluent ECs (passages 1-4) grown in Medium 199supplemented with fetal bovine serum (15%; Gemini, Calabasas, Calif.),human serum (5%, Gemini), endothelial growth supplement (Sigma, St.Louis, Mo.), heparin (90 μg/ml; Sigma) and antibiotics, s described(17). When ECs achieved confluence, experiments were performed byplacing cultures in an environmental chamber (Coy Laboratory Products,Ann Arbor, Mich.) which provided a controlled temperature (37° C.) andatmosphere with the indicated amount of oxygen, carbon dioxide (5%) andthe balance may up of nitrogen. Use of this chamber for cell cultureexperiments has been described previously (15, 18). During exposure ofECs to hypoxia (for a maximum for 16 hours), the oxygen tension in theculture medium was 14-18 torr and there was no change in the medium pH.Reoxygenation was performed by placing ECs in an ambient atmospherecontaining carbon dioxide (5%) at 37° C.

Measurement of Weibel-Palade body exocytosis:

ECs were plated into 24 well plates, rinsed 3 times with Hank's balancedsalt solution, and then exposed to hypoxia or to normoxia for theindicated durations. For experiments in which vWF was measured, cellswere maintained in serum-free medium. All other EC experiments wereperformed in the EC growth medium described above. For measurement ofvMF, 200 μL aliquots of culture supernatant was removed at the indicatedtimes, and a commercially available ELISA (American Diagnostica,Greenwich, Conn.), based on a polyclonal goat anti-human vWF antibody,was performed on duplicate specimens, with a standard curve generatedusing purified human vWF antigen supplied by the same vendor. ECP-selectin expression was determined by measuring the specific bindingof a murine monoclonal anti-human P-selectin antibody (WAPS 12.2 clone,Endogen, Cambridge, Mass.; this is an IgGl which recognizes a calciumsensitive epitope and blocks P-selectin dependent neutrophil adhesion.Antibody was radiolabelled with ¹²⁵I by the lactoperoxidase method (19)using Enzymobeads (Bio-Rad, Hercules, Calif.), stored at 4° C. and usedwithin one week of labelling. Binding assays were performed on HUVECsplated on 96 well plates, in which fresh M199 with 0.1% bovine serumalbumin (Sigma, St. Louis, Mo.) was added immediately prior to eachexperiment. Cells were placed in a humidified environment at 37° C., andexposed to normoxia or H (in the presence or absence of 50 μM probucol,as indicated, Sigma) for the indicated durations. Cell monolayers werefixed for 15 min. with 1% paraformaldehyde¹⁰ (cells exposed to H werefixed while still within the hypoxic environment), visually inspected toensure that the monolayers remained intact, and washed twice with HBSScontaining 0.5% bovine serum albumin (HBSS/A). Monolayers were thenexposed to 10⁵ cpm of ¹²⁵I-labelled anti-P-selectin antibody (WAPS 12.2)in the presence of 200 μg/ml of either unlabelled blocking antibody(WAPS 12.2) or nonblocking anti-P-selectin IgG of the same isotype(anti-GMP-140, AC1.2 clone, Becton-Dickinson, San Jose, Calif.) (20,21). After binding for 1 hours at 37° C., monolayers were washed 4 timeswith HBSS/A, and bound antibody was eluted with 1% triton X-100 in PBS(200 μL/well) and counted. For certain experiments, cycloheximide (10μg/mL, Sigma) was added at the start of the 4 hour normoxic or hypoxicperiod, as indicated. In separate experiments, designed to determine thedegree of inhibition of protein synthesis by cycloheximide treatment,ECs were incubated with methionine- and cysteine-poor minimal essentialmedium (Gibco, Grand Island, N.Y.) in the presence of ³⁵S-methionine and³⁵S-cysteine (either in the presence or absence of cyloheximide, 10μg/mL) (3). Following 4 hours of normoxic exposure, trichloroaceticacid-precipitable material was collected and counted.

Preparation of human PMNs and Measurement of Binding:

In brief, citrated blood from healthy donors was diluted 1:1 with NaCl(0.9%) followed by gradient ultracentrifugation on Ficoll-Hypaque(Pharmacia, Piscataway, N.J.). Following hypotonic lysis of residualerythrocytes (20 sec exposure to distilled H₂O followed byreconstitution with 1.8% NaCl), PMNs were suspended in HBSS with 5 mg/mLof human serum albumin (HBSS/HSA). 50-200×10⁶ PMNs were suspended inHBSS/HSA in the presence of 0.2-0.5 μCi of ¹¹¹Indium oxine (AmershamMediphysics, Port Washington, N.Y.) for 15 minutes at 37° C. Afterwashing with HBSS/HSA, PMNs were gently pelleted (450 g), andresuspended in HBSS/HSA to a final concentration of 5.5×10⁶ PMNs/mL.Following gentle agitation, 100 μL of the radiolabelled PMN suspensionwas added to each well at the indicated time, incubated for 30 minutesat 37° C., and then washed 4 times with HBSS/HSA. Monolayers were thentreated with 1 N NaOH and the contents of each well withdrawn andcounted.

Heterotopic Rat and Mouse Cardiac Transplant Model

Cardiac transplants were performed in the Ono-Lindsey heterotopicisograft model of cardiac transplantation (15, 18, 22). Briefly, maleLewis rats (250-300 grams, Harlan Sprague Dawley, Indianapolis, Ind.)were anesthetized, heparinized, and the donor heart rapidly harvestedfollowing hypothermic high potassium cardioplegic arrest. Hearts werepreserved by flushing the coronary arteries with 4° C. lactated Ringer's(LR) solution (Baxter, Edison, N.J.), sixteen hours of immersion in thesame solution at 4° C., followed by heterotopic transplantation intogender/strain matched recipients, with sequential donor and recipientaortic and donor pulmonary arterial/recipient inferior vena cavalanastomoses performed. Graft survival was assessed by thepresence/absence of cardiac electrical/mechanical activity exactly tenminutes following reestablishment of blood flow, after which the graftwas excised and neutrophil infiltration was quantified bymyeloperoxidase activity, measured as previously described (15, 18). Forcertain experiments, neutrophil depletion of recipient rats wasaccomplished by administering a polyclonal rabbit anti-rat neutrophilantibody (23-25) (Accurate Scientific, Westbury N.Y.) as a singleintravenous injection 24 hours prior to the transplantation procedure.Neutrophil depletion in these animals was confirmed and quantified bycounting remaining neutrophils, identified on Wright-Giemsa stainedsmears of peripheral blood. In other experiments, a blockinganti-P-selectin IgG (250 μg/rat, Cytel, San Diego, Calif.) (13, 14, 26)was administered intravenously 10 minutes prior to the onset ofreperfusion. Murine heart transplants were performed in an identicalfashion using homozyous P-selectin null or wild-type control male micewith a C57BL/6J background (27), with the harvested hearts immediatelyflushed free of native blood with 1.0 mL of 4° C. LR administered down across-clamped aortic root followed by period of hypothermic preservationconsisting of three hours of immersion in lactated Ringer's solution at4° C.

Measurement of vWF in Coronary Effluent from Hypothermically PreservedRat and Human Hearts:

Human coronary sinus samples.

After obtaining informed consent, coronary sinus blood was obtained atthe start and conclusion of routine cardiac surgery in an unselectedseries of 32 patients, with simultaneous sampling of peripheral(arterial) blood in six. Coronary sinus samples were obtained from aretrograde perfusion catheter which was routinely placed in patientsundergoing cardiopulmonary bypass. Plasma samples were centrifuged for 5min at 1500×g to sediment cellular elements, and the plasma aliquotedand frozen at −70° C. until the time of assay. ELISAs were performed forvWF (as described above) and thrombomodulin (Asserchron Thrombomodulin,Diagnostica Stago).

vWF immunoelectrophoresis:

Multimeric composition of the vWF in coronary sinus plasma samples andendothelial cell supernatants was evaluated by performing agarose gelimmunoelectrophoresis. Samples were diluted 1:10, 1:20, and 1:30 (asindicated) and incubated for 30 minutes at 37° C. in Native SampleBuffer (Bio-Rad). Samples (20 μL) were then electrophoresed in a 1.5%agarose gel (0.675 g Low M^(r) agarose, Bio-Rad; 0.045 g SDS; 45 mLTris-Tricine SDS Buffer [Bio-Rad]). Molecular weight markers runsimultaneously on agarose gels were visualized by marking and dividingthe gel, with molecular weight marker locations assigned by Coomasieblue staining. The remaining half of the gel was washed in sodium borate(0.01 M) for 30 minutes followed by overnight electrophoretic transferto a nitrocellulose membrane. The membrane was washed with washingbuffer consisting of tris-buffered saline (pH 7.5) with 0.05% Tween-20,and then blocked for 1 hour with 50 mL of washing buffer containing 2.5g of Carnation instant milk. After rinsing with physiologic saline, themembrane was immersed overnight in washing buffer containing 1 g/dLgelatin and a 1:500 dilution of rabbit anti-human vWF serum (AmericanBioproducts, Parsippany, N.J.). After washing 5 times with washingbuffer, the membrane was immersed for 3 hours with gentle shaking inwashing buffer containing 1 g/dL gelatin and 16.6 μL of boat anti-rabbithorseradish peroxidase conjugated IgG (Bio-Rad), and developed with 65mL of HRP Developer (30 mg HRP Developer powder, Bio-Rad; 10 mLmethanol; 50 mL tris-buffered saline; 50 μL of 30% hydrogen peroxideadded just prior to use).

Statistics.

Analysis of variance was used to compare 3 or more conditions, withpost-hoc comparisons tested using Tukey's procedure. Graft survival datawas analyzed using contingency analysis with the Chi-square statistic.Paired comparison of serial measurements (human CS and peripheral bloodsamples at the start and conclusion of cardiac surgery) were comparedusing Student's t-test for paired variables. Values are expressed asmeans ±SEM, with a p<0.05 considered statistically significant.

Results:

Exposure of cultured ECs to hypoxia results in the release of vWF andtranslocation of P-selectin to the cell surface.

Previous studies have shown that exposure of endothelial cells tohypoxia results in an elevation in intracellular calcium (28). In viewof the association of increased cytosolic calcium with EC Weibel-Paladebody exocytosis in response to thrombin or histamine (29, 30), it wasconsidered whether exposure of ECs to hypoxia could initiate thisprocess. ECs placed in an hypoxic environment (pO₂ 20 torr) releasedmore vWF into the culture supernatants than their normoxic counterparts(FIG. 8A, ELISA; confirmed by immunoelectrophoresis, data not shown).Although a trend towards enhanced levels of vWF was first noted by 1hour of hypoxia, the differences between normoxic and hypoxic vWF levelsdid not become statistically significant until 4 hours of exposure,thereafter increasing steadily for up to 12 hours of observation. Todetermine whether the increased vWF release seen by 4 hours to hypoxiawas due to release of pre-formed vWF, similar experiments were performedin the presence of 10 μg/ml cycloheximide to inhibit protein synthesis.These experiments showed that addition of cycloheximide at the start ofthe hypoxic period decreased hypoxia-induced vWF release by 12.5%,suggesting that the majority of vWF released by hypoxic exposure waspreformed.

Although these experiments were done in their entirety within thehypoxic environment (i.e., there was no reoxygenation), to furtherdemonstrate that this H-mediated exocytosis of Weibel-Palade bodies wasindependent of the formation of reactive oxygen intermediates, theantioxidant probucol (50 μM) was added to the ECs at the onset of H andwas found to have no effect (vWF 4.7±0.31×10⁻³ U/ml at 6 hours of H).The presence of probucol did blunt the further increase in vWF levelsseen following reoxygenation of the hypoxic ECs. The calcium-dependenceof hypoxia-induced Weibel-Palade body exocytosis was demonstrated byexperiments in which ECs were placed in a calcium-free medium at thestart of hypoxic exposure. Absence of extracellular calcium attenuatedH-induced EC release of vWF, and addition of EGTA had an even moresuppressive effect (basal endothelial release of vWF was also diminishedby the reduction of extracellular calcium) (FIG. 8B).

To determine whether hypoxia also induced translocation of P-selectin tothe EC plasmalemmal surface, specific binding of ¹²⁵I-labelled anti-Pselectin IgG to normoxic or hypoxic EC monolayers was examined. Bindingstudies were performed on EC monolayers fixed with paraformaldehydewhile still within the hypoxic environment, to obviate oxygen-freeradical-induced P-selectin expression during reoxygenation. Thesestudies demonstrated enhanced binding of ¹²⁵I-anti-P-selectin IgG byhypoxic compared with normoxic ECs (FIG. 9A). This binding was blockedby unlabelled blocking anti-P-selectin IgG, but not by a nonblockingcontrol anti-P-selectin IgG of the same isotype. Surface expression ofP-selectin was noted at the earliest time points observed (60 minutes ofH), and was observed at similar levels throughout the period of hypoxicexposure (up to 4 hours of observation). It is possible thathypoxia-induced endothelial P-selectin expression was detected at timepoints preceding a statistically significant increase of vWF release insimilarly treated cells, because a portion of the initially secreted vWFbinds tightly to subendothelial matrix (31).

To determine whether protein synthesis was required for hypoxia-inducedP-selectin expression, a separate experiment was performed in whichcycloheximide was given at the onset of normoxia or H, and binding ofradiolabelled anti-P-selectin IgG determined at the 4 hour time point.This experiment demonstrated that even with >85% inhibition of proteinsynthesis (FIG. 9B, Inset), hypoxia still increased endothelialP-selectin expression, albeit at reduced levels (FIG. 9B). To establishthat hypoxia-induced cell-surface P-selectin may participate inneutrophil binding, human neutrophils radiolabelled with ¹¹¹indium oxinewas incubated with hypoxic ECs; enhanced binding to hypoxic monolayerswas observed. Hypoxia-induced ¹¹¹In-PMN binding was blocked by theaddition of a blocking anti-P-selectin IgG, but not by a nonblockinganti-P-selectin IgG (FIG. 9C).

Role of P-selectin dependent neutrophil adhesion in hypothermic/ischemicmyocardial preservation.

To establish the relevance of these observations to hypothermicmyocardial preservation (in which the pO₂ of the preservation solutionwithin the coronary vasculature drops below 20 Torr (15)), hearts wereharvested from male Lewis rats and subjected to hypothermic preservationas described in the methods section. Because neutrophil-mediated damagefollowing cardiac ischemia is well established (32-38), the potentialpathophysiologic role of endothelial P-selectin expression wasinvestigated in an orthotopic rat heart transplant model in whichreperfusion occurred following a period of hypothermic preservation.These experiments showed excellent graft survival and little neutrophilinfiltration if heart transplantation was performed immediatelyfollowing harvest (FIG. 10A, Fresh). However, when similar experimentswere performed with an intervening (16 hour) period of hypothermicpreservation between the harvest and transplantation procedures, therewas a high incidence (90%) of graft failure and marked leukostasis,confirmed histologically and by determining myeloperoxidase activity(FIG. 10A, Prsvd). To demonstrate that neutrophil adhesion wasresponsible, at least in part, for graft failure following prolongedpreservation, transplants were performed following neutrophil depletionof recipient rats. The polyclonal rabbit anti-rat PMN antibody used(23-25) eliminated virtually all circulating PMNs in the recipients (PMNcount 1471±56 vs 67±11 PMNs/mm³ for control and immunodepleted animals,respectively, p<0.001), with little effect on other cell types. When 16hour preserved hearts were transplanted into neutrophil-depletedrecipients to provide a neutrophil-free reperfusion milieu, there was asignificant reduction in graft myeloperoxidase activity and an increasein graft survival (FIG. 10A, Prsvd (−) PMN). Normal recipient ratsinfused with blocking anti-P-selectin IgG 10 minutes prior toreestablishment of blood flow demonstrated a reduction of bothmyeloperoxidase activity as well as improvement in graft survival (FIG.10A, α-PS, Blocking) of a similar magnitude as neutrophil-depletedrecipients. This reduced PMN infiltration and improved graft survivalwas observed despite 16 hours of hypothermic preservation of the donorheart. In sharp contrast, administration of a nonblocking controlantibody (AC1.2) had no beneficial effect on graft leukostasis or graftsurvival (FIG. 10A, α-PS, Non-blocking).

Because in addition to the interactions between ECs and PMNs, plateletsmay also interact with PMNs via a P-selectin-dependent mechanism (39),an experiment was designed to isolate the contribution of endothelialP-selectin to the leukostasis and graft failure which occur followingprolonged hypothermic cardiac preservation. For these experiments, donorhearts from homozygous P-selectin deficient mice could be flushed freeof blood, so that P-selectin null coronary endothelial cells could betransplanted into wild type recipients with P-selectin containingplatelets. Using a murine heterotopic heart transplant model performedidentically to the rat operation, donor hearts were obtained from eitherhomozygous P-selectin null mice (27) or wild-type controls; all heartswere transplanted into wild-type recipients. These experimentsdemonstrated a significantly higher graft survival rate in theP-selectin null→wild type transplants compared with wild type→wild typetransplants (FIG. 10B). This improved graft survival in the formergroups was paralleled by a marked (13-fold) reduction in graftleukostasis (FIG. 10C). Because these hearts had been flushed free ofblood at the start of preservation, these studies implicate coronaryendothelial (rather than platelet-derived) P-selectin in the poorpreservation and leukocyte arrest noted after hypothermic myocardialpreservation.

Weibel-Palade body exocytosis during human cardiac surgery.

To establish the relevance of these findings to humans, the next set ofexperiments were designed to demonstrate that coronary ECs release thecontents of Weibel-Palade bodies during hypothermic cardiac preservationas occurs during routine cardiac surgery. Measurements were made of vWFrelease from the coronary vasculature during a well-defined period ofcardiac ischemia, that which occurs during the period of aorticcross-clamping. Coronary sinus (draining the heart) blood was sampled atthe start (CS₁) and conclusion (CS₂) of aortic cross clamping in 32patients (this interval represents the ischemic period). These patients(23 male, 9 female) had a clinical history of valvular heart disease(n=11) or ischemic heart disease (n=21), and underwent either valverepair/replacement or coronary artery bypass grafting, respectively.Capture ELISAs performed for the integral membrane proteinthrombomodulin (40) demonstrated no change in levels in between the CS₁and the CS₂ samples (4.35±1.2 ng/mL vs 3.48±0.8 ng/mL, p=NS), suggestingthat ECs were not sloughed and cell membrane integrity was maintainedduring cardiac preservation. Similar measurements performed for vWFshowed that there was a consistent and significant increase in vWF thatis secreted during the course of cardiac preservation (0.68±0.06 U/ml vs0.90±0.05 U/ml, CS₁ vs CS₂, p<0.01) (FIG. 11A).

To demonstrate that this vWF was likely to be of coronary endothelialrather than of platelet origin, and hence not simply a consequence ofcardiopulmonary bypass, peripheral blood samples were obtainedsimultaneously with the CS₁ and CS₂ samples, and showed that levels ofvWF were unchanged (0.813±0.52 U/mL vs 0.900±0.41 U/mL, p=NS),suggesting that mechanical perturbation of platelets duringcardiopulmonary bypass was not causative. Because vWF is present inplasma as multimers with a range of M_(r)'s (41-44), with those vWFmultimers from the stimulatable pool (as opposed to those constitutivelysecreted) being of the highest molecular weight (45),immunoelectrophoresis was performed on the CS samples. These gelsdemonstrated that in addition to an overall increases in vWF in the CS₂samples, there appeared to be an increase in high molecular weightmultimers, suggesting release from a stimulatable pool, as is found inendothelial cells (FIG. 11B).

Discussion:

The vasculature plays a critical role in maintaining the extracellularmilieu of organs subjected to ischemia and reperfusion, a role which ischiefly orchestrated by the ECs lining the endovascular lumen. The ECresponds to a period of oxygen deprivation by striking phenotypicmodulation, becoming prothrombotic (46) and proinflammatory (1,4,6). ECsexposed to hypoxia secrete the proinflammatory cytokines IL-1 (4) andIL-8 (6) which may serve to direct leukocyte traffic to areas ofischemia. Because these processes require de novo protein synthesis,they do not explain the immediate events which occur following a periodof hypothermic preservation. While enhanced expression of ICM-1 andinduction of E-selectin may contribute at later times to leukocytearrest in cardiac grafts, this does not explain the rapid leukostasisobserved following cold preservation, in which protein synthesis islikely to be considerably slowed. In this context, cycloheximidepre-treatment does not alter the early (90-120 minute) PMN adhesion seenfollowing hypoxic exposure of ECs (7), suggesting that de novo proteinsynthesis need not be involved in hypoxia-mediated increases in PMNbinding. Although platelet activating factor (PAF) may participate inhypoxia-mediated PMN adhesion (7,47) and activation (48,49), PAF is notstored and must be synthesized, which may lessen its importance duringthe hypothermic period during myocardial preservation. It is for thisreason that rapid EC expression of pre-formed P-selectin fromsubplasmalemmal storage sites in Weibel-Palade bodies (9, 50, 51) mayrepresent the most important mechanism for early PMN recruitmentfollowing hypothermic preservation. Weibel-Palade bodies are found inabundance within the coronary microvasculature (52), suggesting theirparticular importance in cardiac preservation.

The data show Weibel-Palade exocytosis occurs both in response tohypoxia per se, as well as in human hearts during hypothermicpreservation. While it is difficult to precisely identify an endothelialorigin for the vWF observed in the human coronary sinus samples, studiesof platelets following cardiopulmonary bypass demonstrate no increase insurface P-selectin expression or α-granule secretion (53, 54). Thissuggests that the observed increase in coronary sinus vWF followingaortic cross-clamping is not of platelet origin. Two aspects of the dataalso suggest that the vWF released following ischemia is of endothelialorigin; (1) Peripheral vWF levels remains unchanged while coronary sinuslevels are increased following myocardial ischemia, suggesting that theelevated vWF was emanating from the heart, not the cardiopulmonarybypass apparatus; (2) The transgenic, P-selectin null donor hearts wereflushed free of donor blood at the onset of preservation, so that whentransplanted into wild-type recipients, presumably coronary endothelial(not platelet) P-selectin is absent. These experiments demonstrate theimportant contribution of endothelial P-selectin to the neutrophilrecruitment which accompanies reperfusion.

It is not surprising that P-selectin should be important followinghypothermic myocardial preservation, recent studies have demonstratedthat P-selectin is an important mediator of neutrophil-inducedreperfusion damage following normothermic ischemia, as has been shown inrabbit ear (26) and feline cardiac ischemia (14) models. Becauseoxidants cause expression of P-selectin at the EC surface (10), it wasimportant in these studies to evaluate the role of the hypoxic periodalone as it may prime ECs to recruit the first wave at PMNs, withfurther PMN recruitment amplified with the onslaught of reaction oxygenintermediates produced in the reperfusion microenvironment. Although onereport has suggested that hypoxia might induce EC P-selectin expression,these experiments (7) were actually performed following reoxygenation, acondition which is known to induce both superoxide (18, 55) andneutrophil adherence to cultured ECs (56). By contrast, the experimentsdescribed herein were performed entirely within a hypoxic environment tocompletely prevent the possibility of reoxygenation, and antioxidantsfailed to block hypoxia-induced P-selectin expression, suggesting thatthe observations described herein reflect hypoxia hypoxia per se ratherthan reoxygenation. Furthermore, the cardiac protection demonstratedherein using a strategy whereby blood-free preserved hearts fromtransgenic P-selectin null mice are transplanted into recipients withwild-type platelets demonstrates that endothelial P-selectin expressioncan be deleterious following hypothermic cardiac preservation. BecauseWeibel-Palade body exocytosis occurs during hypothermic cardiacpreservation in humans, these studies suggest that myocardialpreservation may be enhanced by therapeutic strategies designed to blockthe activity of P-selectin expressed at the endothelial surface.

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EXAMPLE 3 Procedural and Strain-Related Variables Significantly EffectOutcome in a Murine Model of Focal Cerebral Ischemia

The recent availability of transgenic mice has led to a burgeoningnumber of reports describing the effects of specific gene products onthe pathophysiology of stroke. Although focal cerebral ischemia modelsin rats have been well-described, descriptions of a murine model ofmiddle cerebral artery occlusion are scant, and sources of potentialexperimental variability remain undefined. It was hypothesized thatslight technical modifications would result in widely discrepant resultsin a murine model of stroke, and that controlling surgical andprocedural conditions could lead to reproducible physiologic andanatomic stroke outcomes. To test this hypotheses, a murine model wasestablished which would permit either permanent or transient focalcerebral ischemia by intraluminal occlusion of the middle cerebralartery (MCA). This study provides a detailed description of the surgicaltechnique, and reveals important differences between strains commonlyused in the production of transgenic mice. In addition to strain-relateddifferences, infarct volume, neurologic outcome, and cerebral blood flowappear to be importantly affected by temperature during the ischemic andpost-ischemic periods, mouse size, and size of the suture whichobstructs the vascular lumen. When these variables were kept constant,there was remarkable uniformity of stroke outcome. These data emphasizethe protective effects of hypothermia in stroke, and should help tostandardize techniques among different laboratories to provide acohesive framework for evaluating the results of future studies intransgenic animals.

Introduction

The recent advent of genetically altered mice provides a uniqueopportunity to evaluate the role of single gene products in thepathophysiology of stroke. Although there is an increasing number ofreports about the effect of cerebral ischemia in transgenic mice, todate, there exists no detailed description of the murine modelsinvolved, nor is there a detailed analysis of potentially importantprocedural variables which may effect stroke outcome. Most descriptionsof a murine model (1,4,8,9,14,17-19,23,24) are devolved descriptions ofthe widely used rat models of focal cerebral ischemia (22,26). Althoughthere has been some attention paid to strain related differences in thesusceptibility of mice to cerebral ischemia (4), few technicalconsiderations have been addressed in published studies. Because pilotdata demonstrated that minor differences in operative procedures orpostoperative care translated into major differences in stroke outcome,the current study was undertaken to systematically identify importantsurgical, technical, and anatomic considerations required to obtainconsistent results in a murine model of focal cerebral ischemia. Whenstokes are created in a rigidly controlled manner, differences, due tothe absence (or overexpression) of a single gene product, should bereadily discernable.

This study presents a detailed rendering of a reproducible murine modelof focal cerebral infarction based on modifications of the original ratmodel (26). This study identifies procedural variables that have a largeimpact on stroke outcome which have not been previously reported intechnical descriptions of murine stoke models. These variables includesuture length and gauge, methods of vascular control., temperatureregulation in mice, and differences between strains commonly used in thebreeding of transgenic animals. As the model described lends itself tothe study of either permanent or transient focal cerebral ischemia,evidence is presented that with carefully chosen ischemia times, infarctvolume and mortality in reperfused animals can be made to approximatethose seen with permanent occlusion. Understanding potentialmodel-dependent sources of variability in stroke outcome can help toclarify divergent results between different laboratories. Adoption of astandardized model which yields consistent results is an important firststep towards the use of transgenic mice in the study of thepathophysiology of stroke.

Materials and Methods

Animal Purchase and Anesthesia

Male mice of three different strains (C57 BlackJ6, CD-1 and 129J) werepurchased from Jackson Laboratories (Bar Harbor, Me.). Animals wereeight to ten weeks of age and weighed between 18-37 grams (as indicated)at the time of experiments. Mice were anesthetized with anintraperitoneal injection of 0.3 ml of ketamine (10 mg/cc) and xylazine(0.5 mg/cc). An additional dose of 0.1 cc was given prior to withdrawalof the catheter in animals undergoing transient ischemia. On the dayfollowing surgery, anesthesia was repeated immediately prior to laserdoppler flow measurement and humane euthansia. These procedures havebeen approved by the Institutional Animal Care and Use Committee atColumbia University, and are in accordance with AALAC guidelines for thehumane care and use of laboratory animals.

Surgical Set-up

The animal was positioned supine on a gauze pad which rests on atemperature controlled operating surface (Yellow Springs Instruments,Inc. [YSI], Yellow Springs, Ohio). A rectal temperature probe (YSI) wasinserted, in order to regulate the temperature of the operating surfaceto maintain a constant animal core temperature of 36-38° C. Tofacilitate exposure, the right hindpaw and left forepaw were taped tothe operating surface, the right forepaw was taped to the animal'schest, and the tail was taped to the rectal probe (FIG. 12A). A midlineneck incision was made by gently lifting the loose skin between themanubrium and the jaw and excising a 1 cm² circle of skin. The pairedmidline submandibular glands directly underlying this area were bluntlydivided, with the left gland left in situ. The right gland was retractedcranially with an small straight Sugita aneurysm clip (Mizutto America,Inc., Beverly, Mass.) secured to the table by a 4.0 silk and tape. Thesternocleidomastoid muscle was then identified, and a 4.0 silk ligatureplace around its belly. This ligature was drawn inferolaterally, andtaped to the table, to expose the omohyoid muscle covering the carotidsheath. The exposure is shown in FIG. 12B.

Operative Approach

Once the carotid sheath was exposed, the mouse and the temperaturecontrol surface were placed under an operating microscope (16-25 X zoom,Zeiss, Thornwood, N.Y.), with a coaxial light source used to illuminatethe field. Under magnification, the omohyoid muscle was carefullydivided with pickups. The common carotid artery (CCA) was carefullyfreed from its sheath, taking care not to apply tension to the vagusnerve (which runs lateral to the CCA). Once freed, the CCA was isolatedwith a 4.0 silk, taped loosely to the operating table. Once proximalcontrol of the CCA was obtained, the carotid bifurcation was placed inview. The occipital artery, which arises from the proximal externalcarotid artery and courses postero-laterally across the proximalinternal carotid artery (ICA) to enter the digastric muscle, wasisolated at its origin, and divided using a Malis bipolarmicorcoagulator (Codman-Schurtleff, Randolph, Mass.). This enabledbetter visualization of the ICA as it courses posteriorly and cephaladunderneath the stylohyoid muscle towards the skull base. Just before theICA enters the skull it gives off a pterygopalatine branch, whichcourses laterally and cranially. This branch was identified, isolated,and divided at its origin, during which time the CCA-ICA axisstraightens. A 4.0 silk suture was then placed around the internalcarotid artery for distal control, the end of which was loosely taped tothe operating surface.

Next, the external carotid artery was placed in view. Its cranio-medialcourse was skeletonized and its first branch, the superior thyroidartery, was cauterized and divided. Skeletonization was subsequentlycarried out distally by elevation of the hyoid bone to expose theartery's bifurcation into the lingual and maxillary arteries. Justproximal to this bifurcation the external carotid was cauterized anddivided. Sufficient tension was then applied to the silk suturessurrounding the proximal common, and distal internal, carotid arteriesto occlude blood flow, with care taken not to traumatize the arterialwall. Tape on the occluding sutures was readjusted to maintainocclusion.

Introduction and Threading of the Occluding Intraluminal Suture

Immediately following carotid occlusion, and arteriotomy was fashionedin the distal external carotid wall just proximal to the cauterizedarea. Through this arteriotomy, a heat-blunted 5.0 or 6.0 nylon suture(as indicated in the Results section) was introduced (FIGS. 12C and12D). As the suture was advanced to the level of the carotidbifurcation, the external stump was gently retracted caudally directingthe tip of the suture into the proximal ICA. Once the occluding sutureentered the ICA, tension on the proximal and distal control sutures wasrelaxed, and the occluding suture was slowly advanced up the ICA towardsthe skull base under direct visualization (beyond the level of the skullbase, sight of the occluding suture is lost). Localization of the distaltip of the occluding suture across the origin of the middle cerebralartery (MCA) (proximal to the origin of the anterior cerebral artery)was determined by the length of suture chosen (12 mm or 13 mm adindicated in the Results section, shown in FIG. 12C), by laster dopplerflowmetry (see Ancillary physiological procedures section), and bypost-sacrifice staining of the cerbral vasculature (see below). Afterplacement of the occluding suture was complete, the external carotidartery stump was cauterized to prevent bleeding through the arteriotomyonce arterial flow was reestablished.

Completion of Surgical Procedure

For all of the experiments shown, the duration of carotid occlusion wasless then two minutes. To close the incision, the sutures surroundingthe proximal and distal CCA, as well as the sternocleidomastoid muscle,were cut and withdrawn. The aneurysm clip was removed from thesubmandibular gland and the gland was laid over the operative field. Theskin edges were then approximated with one surgical staple and theanimal removed from the table.

Removal of the Occluding Suture to Establish Transient Cerebral Ischemia

Transient cerebral ischemia experiments required reexploration of thewound to remove the occluding suture. For these experiments, initialwound closure was performed with a temporary aneurysm clip rather than asurgical staple to provide quick access to the carotid. Proximal controlwith a 4-0 silk suture was reestablished prior to removal of theoccluding suture to minimize bleeding from the external carotid stump.During removal of the occluding suture, cautery of the external carotidartery stump was begun early, before the distal suture has completelycleared the stump. Once the suture was completely removed, the stump ismore extensively cauterized. Reestablishment of flow in the extracranialinternal carotid artery was confirmed visually and the wound was closedas for permanent focal ischemia described above. Confirmation ofintracranial reperfusion was accomplished with laser doppler flowmetry(see Ancillary physiological procedures section).

Calculation of Stroke Volume

Twenty-four hours after middle cerebral artery occlusion, surviving micewere reanesthetized with 0.3 cc of ketamine (10 mg/ml) and xylazine (0.5mg/ml). After final weights, temperatures and cerebral blood flowreadings were taken (as described below), animals were perfused with 5ml of a 0.15% solution of methylene blue and saline to enhancevisualization of the cerebral arteries. Animals were then decapitated,and the brains were removed. Brains were then inspected for evidence ofcorrect catheter placement, as evidenced by negative staining of thevascular territory subtended by the MCA, and placed in a mouse brainmatrix (Activational Systems Inc., Warren, Mich.) for 1 mm sectioning.Sections were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC)in 0.9% phosphate-buffered saline, incubated for 30 minutes at 37° C.,and placed in 10% formalin (5). After TTC staining, infarcted brain wasvisualized as an area of unstained (white) tissue in a surroundingbackground of viable (brick red) tissue. Serial sections werephotographed and projected on tracing paper at a uniform magnification;all serial sections were traced, cut out, and the paper weighed by atechnician blinded to the experimental conditions. Under theseconditions, infarct volumes are proportional to the summed weights ofthe papers circumscribing the infarcted region, and were expressed as apercentage of the right hemispheric volume. These methods have beenvalidated in previous studies (3,12,15,16).

Ancillary Physiological Studies

Ancillary physiogical studies were performed on each of the threedifferent strains used in the current experiments, immediately prior toand after the operative procedure. Systemic blood pressures wereobtained by catheterization of the infrarenal pressures were obtained bycatheterization of the infrarenal abdominal aorta, and measured using aGrass Model 7 polygraph (Grass Instrument Co., Quincy, Mass.). Anarterial blood sample was obtained from this infrarenal aortic catheter;arterial pH, pCO₂ (mm Hg), pO₂ (mm Hg) and hemoglobin oxygen saturation(%) were measured using a Blood Gas Analyser and Hemoglobinometer (GrassInstrument Co., Quincy, Mass.). Because of the need for arterialpuncture and abdominal manipulation to measure these physiologicparameters, animals were designated solely for these measurements(stroke volumes, neurologic outcome, and cerebral blood flows were notmeasured in these same animals).

Transcranial measurements of cerebral blood flow were made using laserdoppler flowmetry (Perimed, Inc., Piscataway, N.J.) after reflections ofthe skin overlying the calvarium, as previously described (10)(transcranial readings were consistently the same as those made aftercraniectomy in pilot studies). To accomplish these measurements, animalswere placed in a stereotactic head frame, after which they underwentmidline skin incision from the nasion to the superior nuchal line. Theskin was swept laterally, and a 0/7 mm straight laser doppler probe(model #PF2B) was lowered onto the cortical surface, wetted with a smallamount of physiologic saline. Readings were obtained 2 mm posterior tothe bregma, both 3 mm and 6 mm to each side of midline using asterotactic micromanipulator, keeping the angle of the probeperpendicular to the cortical surface. Relative cerebral blood flowmeasurements were made immediately after anesthesia, after occlusion ofthe MCA, and immediately prior to euthanasia, and are expressed as theratio of the doppler signal intensity of the ischemic compared with thenonischemic hemisphere. For animals subjected to transient cerebralischemia, additional measurements were made just before and just afterwithdrawal of the suture, initiating reperfusion.

The surgical procedure/intraluminal MCA occlusion was considered to betechnically adequate if ≧50% reduction in relative cerebral blood flowwas observed immediately following placement of the intraluminaloccluding catheter (15 of the 142 animals used in this study [10.6%]were exluded due to inadequate drop in blood flow at the time ofocclusion). These exclusion criteria were shown in preliminary studiesto yield levels of ischemia sufficient to render consistent infarctvolumes by TTC staining. Reperfusion was considered to be technicallyadequate if cerebral blood flow at catheter withdrawal was at leasttwice occlusion cerebral blood flow (13/17 animals in this study [76%]).

Temperature

Core temperature during the peri-infarct period was carefully controlledthroughout the experimental period. Prior to surgery, a baseline rectaltemperature was recorded (YSI Model 74 Thermistemp rectal probe, YellowSprings Instruments, Inc., Yellow Springs, Ohio). Intraoperatively,temperature was controlled using a thermocouple-controlled operatingsurface. Following MCA occlusion, animals were placed for 90 minutes inan incubator, with animal temperature maintained at 37° C. using therectal probe connected via thermocouple to a heating source in theincubator. Temperature was similarly controlled in those animalssubjected to transient ischemia, including a 45 minute (ischemic) periodas well as a 90 minute post-ischemic period in the incubator. Followingplacement in the core-temperature incubator, animals were returned totheir cages for the remaining duration of pre-sacrifice observation.

Neurological Exam

Prior to giving anesthesia at the time of euthanasia, mice were examinedfor obvious neurological deficit using a four-tiered grading system: (1)normal spontaneous movements, (2) animal circling towards the right, (3)animal spinning to the right, (4) animal crouched on all fours,unresponsive to noxious stimuli. This system was shown in preliminarystudies to accurately predict infarct size, and is based on systemsdeveloped for use in rats (6).

Data Analysis

Stroke volumes, neurologic outcome scores, cerebral blood flows andarterial blood gas data were compared using an unpaired Student'st-test. Values are expressed as means +SEM, with a p<0.05 consideredstatistically significant. Mortality data, where presented was evaluatedusing chi-squared analysis.

Results

Effects of Strain

Three different commonly used mouse strains (CD1, C57/B16, and 129J)were used to compare the variability in stroke outcome followingpermanent focal cerebral ischemia. To establish that there were no grossanatomic differences in collateralization of the cerebral circulation,the Circle of Willis was visualized using India ink in all three strains(FIG. 13). These studies failed to reveal any gross anatomicdifferences. Mice of similar sizes (20+0.8 g, 23+0.4 g, and 23+0.5 g for129J, CD1, and C57B1 mice, respectively) were then subjected topermanent focal ischemia under normothermic conditions using a 12 mmlength of 6-0 nylon occluding suture. Significant strain-relateddifferences in infarct volume were noted, with infarcts in 129J micebeing significantly smaller than those observed in CD1 and C57/B16 micedespite identical experimental conditions (FIG. 14A). Differences ininfarct size were paralleled by neurological exam, with the highestscores (i.e., most severe neurologic damage) being seen in the C57/B16and CD1 mice (FIG. 14B).

To determine the relationship between infarct volume and cerebral bloodflow to the core region, laser doppler flowmetry was performed throughthe thin murine calvarium. No preoperative strain-related differences incerebral blood flow were observed, corresponding to the lack of grossanatomic differences in vascular anatomy (FIG. 13). Measurement ofcerebral blood flow immediately following insertion of the occludingcatheter revealed that similar degees of flow reduction were created bythe procedure (the percentage of ipsilateral/contralateral flowimmediately following insertion of the obstructing catheter was 23+2%,19+2%, 17+3% for 129J, CD1, and C57/B16 mice, respectively). Notsurprisingly, blood flow to the core region measured at 24 hours justprior to euthanasia demonstrated the lowest blood flows in those animalswith the most severe neurologic injury (FIG. 14C).

Anatomic and Physiologic Characteristics of Mice

Baseline arterial blood pressure, as well as arterial blood pressuresfollowing middle cerebral artery occlusion, were nearly identical forall animals studied, and were not effected by mouse strain or size(Table I). Analysis of arterial blood for pH, pCO₂, and hemoglobinoxygen saturation (%) similarly revealed no significant differences(Table I).

Effect of Animal Size and Bore of the Occluding Suture

To investigate the effects of mouse size on stroke outcome, mice of twodifferent sizes (23+0.4 g and 31+0.7 g) were subjected to permanentfocal cerebral ischemia. To eliminate other potential sources ofvariability in these experiments, experiments were performed undernormothermic conditions in mice of the same strain (CD1), usingoccluding sutures of identical length and bore (12 mm 6-0 nylon). Underthese conditions, small mice (23+0.4 g) sustained consistently largeinfarct volumes (28+9% of ipsilateral hemisphere). Under identicalexperimental conditions, large mice (31+0.7 g) demonstrated much smallerinfarcts (3.2+3%, p=0.02, FIG. 15A), less morbidity on neurological exam(FIG. 15B), and a tendency to maintain higher ipsilateral cerebral bloodflow following infarction than smaller animals (FIG. 15C).

Because it was hypothesized that the reduction in infarct size infarctsin these large animals was related to a mismatch in diameter/lengthbetween occluding suture and the cerebral blood vessels, longer/thickeroccluding sutures were fabricated (13 mm, 5-0 nylon ) for use in theselarger mice. Large CD1 mice (34+0.8 g) which underwent permanentocclusion with these larger occluding sutures sustained a markedincrease in infarct volumes (50+10% of ipsilateral hemisphere, p<0.0001compared with large mice infarcted with the smaller occluding suture,FIG. 15A). These larger mice infarcted with larger occluding suturesdemonstrated higher neurologic deficit scores (FIG. 15B) and loweripsilateral cerebral blood flows (FIG. 15C) compared with similarlylarge mice infarcted with smaller occluding sutures.

Effects of Temperature

To establish the role of perioperative hypothermia on the stroke volumesand neurologic outcomes following MCA occlusion, small C57/B16 mice(22+0.4 g) were subjected to permanent MCA occlusion with 12 mm 6-0gauge suture, with normothermia maintained for two different durations;Group 1 (“Normothermia”) was operated as described above, maintainingtemperature at 37° C. from the preoperative period until 90 minutespost-occlusion. Group 2 animals (“Hypothermia”) were maintained at 37°C. from preop to only 10 minutes post-occlusion, as has been describedpreviously (14). Within 45 minutes following removal from thethermocouple-controlled warming incubator, core temperature in thissecond group of animals dropped to 33.1+0.4° C. (and dropped further to31.3+0.2° C. at 90 minutes). Animals operated under conditions ofprolonged normothermia (Group 1) exhibited larger infarct volumes(32+9%) than hypothermic (Group 2) animals (9.2+5%, p=0.03, FIG. 16A).Differences in infarct volume were mirrored by differences inneurological deficit (3.2+0.4 vs. 2.0+0.8, p=0.02, FIG. 16B), but werelargely independent of cerebral blood flow (52+5 vs. 52+7, p=NS, FIG.16C).

Effects of Transient MCA Occlusion

Because reperfusion injury has been implicated as an important cause ofneuronal damage following cerebrovascular occlusion (25), a subset ofanimals was subjected to a transient (45 minute) period of ischemiafollowed by reperfusion as described above, and comparison s made withthose animals which underwent permanent MCA occlusion. The time ofocclusion was chosen on the basis of preliminary studies (not shown)which demonstrated unacceptibly high mortality rates (>85%) with 180minutes of ischemia and rare infarction (<15%) with 15 minutes ofischemia. To minimize the confounding influence of other variables,other experimental conditions were kept constant (small (22.5+0.3 g)C57/B16 mice were used, the occluding suture consisted of 12 mm 6-0nyon, and experiments were performed under normothermic conditions). Theinitial decline in CBF immediately post-occlusion were similar in bothgroups (16+2% vs 17+3%, for transient vs permanent occlusion groups,respectively, p=NS). Reperfusion was confirmed both by laser doppler(2.3-fold increase in blood flow following removal of the occludingsuture to 66+13%), and visually by intracardiac methylene blue dyeinjection in representative animals. Infarct sizes (29+10% vs. 32+9%),neurologic deficit scores (2.5+0.5 vs. 3.2+0.4), and sacrifice cerebralblood flow (46+18% vs. 53+5%) were quite similar between between animalssubjected to transient cerebral ischemia and reperfusion and thosesubjected to permanent focal cerebral ischemia (p=NS, for all groups)FIGS. 17A-17C).

Discussion

The growing availability of genetically altered mice has led to anincreasing use of murine models of focal cerebral ischemia to imputespecific gene products in the pathogenesis of stroke. Although recentpublications describe the use of an intraluminal suture to occlude themiddle cerebral artery to create permanent and/or transient cerebralischemia in mice, there has been only scant description of the necessarymodifications of the original technical report in rats(8,14,17-19,24,26). The experiments described herein not only provide adetailed technical explanation of a murine model suitable for eitherpermanent or transient focal middle cerebral artery ischemia, but alsoaddress potential sources of variability in the model.

Importance of Strain

One of the most important potential sources of variability in the murinecerebral ischemia model described herein is related to the strain ofanimal used. The data suggest that, of the three strains tested, 129Jmice are particularly resistant to neurologic injury following MCAocclusion. Although Barone similarly found differences in stroke volumesbetween 3 strains of mice (BDF, CRW and BALB/C), these differences wereascribed to variations in the posterior communicating arteries in thesestrains (4). As anatomical differences in cerebrovascular anatomy werenot grossly apparent in the study (FIG. 13), the data suggests thatnon-anatomic strain-related differences are also important in outcomefollowing MCA occlusion.

As stroke outcome differs significantly between 2 strains of mice (129Jand C57/B16) commonly used to produce transgenic mice via homologousrecombination in embryonic stem cells (11), the data suggest animportant caveat to experiments performed with transgenic mice. Becauseearly founder progeny from the creation of transgenic animals with thesestrains have a mixed 129J / C57/B16 background, ideally experimentsshould be performed either with sibling controls or after a sufficientnumber of backcrossings to ensure strain purity.

Importance of Size

Larger animals require a longer and thicker intralumenal suture tosustain infarction volumes which are consistent with those obtained insmaller animals with smaller occluding sutures. Size matching of animaland suture appear to be important not only to produce consistentcerebral infarction, but whereas too small a suture leads toinsufficient ischemia, too large a suture leads to frequentintracerebral hemorrhage and vascular trauma (unpublished observation).

The use of animals of similar size is important not only to minimizepotential age-related variability in neuronal susceptability to ischemicinsult, but also to ensure that small differences in animal size do notobfuscate meaningful data comparison. In this example, it isdemonstrated that size differences of as little as 9 grams can have amajor impact on infarct volume and neurologic outcome following cerebralischemia. Further experiments using larger bore occluding suture inlarger animals suggest that the increased propensity of smaller animalsto have larger strokes was not due to a relative resistance of largeranimals to ischemic neuronal damage, but was rather due to small size ofthe suture used to occlude the MCA in large animals. Although these datawere obtained using CD1 mice, similar studies have been performed andfound these results to be true with other mouse strains as well, such asC57/B16 (unpublished data). Previously published reports use mice ofmany different sizes (from 21 g to 35 g), as well as different suturediameters and lengths which are often unreported (14,17). The studiesindicate that animal and suture size are important methodological issueswhich must be addressed in scientific reports.

Importance of Temperature

It has long been recognized that hypothermia protects a number of organsfrom ischemic injury, including the brain. Studies performed in ratshave demonstrated that intraischemic hypothermia up to 1 hour post-MCAocclusion is protective (2,15), reducing both mortality and infarctvolumes with temperatures of 34.5 degrees. Although these results havebeen extrapolated to murine models of cerebral ischemia in that studiesoften describe maintenance of normothermia in animals, the post-MCAocclusion temperature monitoring periods have been extremely brief(“immediately after surgery” or “10 minutes after surgery”) (4,14). Theresults indicate that animals fail to autoregulate their temperaturebeyond these brief durations, becoming severely hypothermic during thepostoperative period, and that temperature differences up to 90 minutesfollowing MCA occlusion can have a profound effect on indices of strokeoutcome following MCA occlusion (longer durations of normothermia werenot studied). While others have ensured normothermia using a feedbacksystem based on rectal temperature similar to the one described herein,the duration of normothermia is often not specified (17). The resultsargue for clear identification of methods for monitoring and maintainingtemperature, as well as the durations involved, so that experimentalresults can be compared both within and between Centers studying thepathophysiology of stroke.

Transient vs Permanent Occlusion

The pathophysiology of certain aspects of permanent cerebral ischemiamay well be different form that of cerebral ischemia followed byreperfusion, so it was important that a model be described whichpermitted analysis of either condition. Although differences betweenthese two models were not extensively tested in the current series ofexperiments, under the conditions tested (45 minutes of ischemiafollowed by 23 hours of reperfusion), no significant differences werefound in any index of stroke outcome. Variable durations of ischemia andreperfusion have been reported in other murine models of transientcerebral ischemia, with ischemic times ranging from 10 minutes to 3hours and reperfusion times ranging from 3 to 24 hours (17,24). Studiesin rats have shown that short periods of ischemia followed byreperfusion are associated with smaller infarcts than permanentocclusion (21,25). However as the duration of ischemia increases beyonda critical threshold (between 120 and 180 minutes), reperfusion isassociated with larger infarcts (7,21,26). For the current series ofexperiments, the durations of ischemia and reperfusion were chosen so asto obtain infarcts comparable to those observed following permanent MCAocclusion, which is likely to explain why the data failed to showdifferences between permanent and transient ischemia. These durations inthe transient model were chosen after pilot experiments revealed thatshorter ischemic durations (15 minutes) rarely led to infarction,whereas 180 minutes of occlusion followed by reperfusion led to massiveinfarction and nearly 100% mortality within 4-6 hours in normothermicanimals (unpublished observation). Although indices of stroke outcomemay be measured earlier than 24 hours, the 24 hour observation time waselected because observation at this time permits the study of delayedpenumbral death, which is likely to be clinically relevant to thepathophysiology of stroke in humans. Furthermore, 24 hours has beenshown in a rat model to be sufficient for full infarct maturation(3,12,15,16)

Technical Aspects of the Murine Model

Technical aspects of the surgery needed to create focal cerebralischemia in mice differ in certain important respects from that in rats.Self-retaining retractors, which have been advocated in previous reportsin rats 26), are unwieldy in mice. Suture-based retraction secured withtape provides a superior alternative. In rats, clip occlusion of theproximal and distal carotid artery after mobilization of the externalcarotid artery has been reported (26), but creates more carotid traumaand hemmorhage in mice. Without distal internal carotid control, whichhas not been previously described in mice, backbleeding from theexternal carotid artery is consistently uncontrollable. Using thetechniques described in this paper, surgery can be completed withvirtually no blood loss, which is especially important given the smallblood volume in mice.

Unlike the rat model, the occlusion and transection of the externalcarotid artery branches and the pterygopalatine artery in the murinemodel is achieved with electrocautery alone. Previous reports of murinesurgery have been unclear as to whether or not the pterygopalatineartery was taken (17,24). Others have described a method with permanentocclusion of the common carotid artery and trans-carotid insertion ofthe suture without attention to either the external carotid system orthe pterygopalatine artery. While effective for permanent occlusion,this latter method makes reperfusion studies impossible.

The method of reperfusion originally described in the rat requires blindcatheter withdrawal without anesthesia (26). When attempted in pilotstudies in mice, several animals hemorrhaged. Therefore, a method ofsuture removal under direct visualization in the anesthetized animal wasdeveloped, which not only allows visual confirmation of extracranialcarotid artery reperfusion, but also affords meticulous hemostasis.Further, the method permits immediate pre- and post-reperfusion laserdoppler flowmetry readings in the anesthetized animal.

These laser doppler flowmetry readings are similar to those described byKamii et al. and Yang et al. in that the readings are madeintermittantly and with the use of a stereotactic micromanipulator(17,24). The readings differ, however, in that the coordinates used (2mm posterior and 3 and 6 mm lateral to the bregma) are slightly morelateral and posterior than the previously published core and penumbralcoordinates (1 mm posterior and 2 mm and 4.5 mm lateral to the bregma).These coordinates, which were adopted based on pilot studies, are thesame as those used by Huang et al (14).

Conclusion

These studies demonstrate specific technical aspects of a murine modelof focal cerebral ischemia and reperfusion which permits reproducibilityof measurements between different laboratories. In addition, thesestudies provide a framework for understanding important proceduralvariables which can greatly impact on stroke outcome, which should leadto a clear understanding of non-procedure related differences underinvestigation. Most importantly, this study points to the need forcareful control of mouse strain, animal and suture size, and temperaturein experimental as well as control animals. Conditions can beestablished so that stroke outcome is similar between models ofpermanent focal cerebral ischemia and transient focal cerebral ischemia,which should facilitate direct comparison and permit the study ofreperfusion injury. The model described in this study should provide acohesive framework for evaluating the results of future studies intransgenic animals, to facilitate an understanding of the contributionof specific gene products in the pathophysiology of stroke.

Table I

Pre- and post-operative physiologic parameters. MAP, mean arterialpressure; pCO₂, partial pressure of arterial CO₂ (mm Hg); O₂ Sat,saturation (%); Hb, hemoglobin concentration (g/dl); Preoperative,anesthetized animals prior to carotid dissection; Sham, anesthetizedanimals undergoing the surgical described in the text, immediately priorto introduction of the occluding suture; Stroke, anesthetized animalsundergoing the surgical described in the text, immediately afterintroduction of the occluding suture. p=NS for all between-groupcomparisons. (data shown is for small 22 gram C57/B16 mice).

PARAMETER PREOPERATIVE SHAM STROKE MAP 102 ± 5.5  94 ± 1.9  88 ± 4.9 pH7.27 ± 0.02 7.23 ± 0.04 7.28 ± 0.01 pCO₂  46 ± 1.3  44 ± 1.3  47 ± 3.5O₂ Sat  89 ± 1.6  91 ± 1.8  85 ± 2.2 Hb 14.6 ± 0.42 14.3 ± .12  14.2 ±0.12

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EXAMPLE 4 Exacerbation of Cerebral Injury In Mice Which Express theP-Selectin Gene: Identication of P-selectin Blockade as a New Target forthe Treatment of Stroke

There is currently a stark therapeutic void for the treatment ofevolving stroke. Although P-selectin is rapidly expressed by hypoxicendothelial cells in vitro, the functional significance of P-selectinexpression in stroke remains unexplored. In order to identify thepathophysiological consequences of P-selectin expression and to identifyP-selectin blockade as a potential new approach for the treatment ofstroke, experiments were performed using a murine model of focalcerebral ischemia and reperfusion. Early P-selectin expression in thepost-ischemic cerebral cortex was demonstrated by the specificaccumulation of radiolabelled anti-murine P-selectin IgG. In parallelexperiments, neutrophil accumulation in the ischemic cortex of miceexpressing the p-selectin gene (PS +/+) was significantly greater thatthat demonstrated in homozygous P-selectin null mice (PS −/−). Reducedneutrophil influx was accompanied by greater postischemic cerebralreflow (measured by laser doppler) in the PS −/− mice. In addition, PS−/− mice demonstrated smaller infarct volumes (five-fold reduction,p<0.05). and improved survial compared with PS+/+ mice (88% vs. 44%,p<0.05). Functional blockade of P-selectin in PS +/+ mice using amonoclonal antibody directed against murine P-selectin also improvedearly reflow and stroke outcome compared with controls. These data arethe first to demonstrate a pathophysiological role for P-selectin instroke, and suggest that P-selectin blockade may represent a newtherapeutic target for the treatment of stroke.

Introduction

Ischemic stroke constitutes the third leading cause of death in theUnited States today¹. Until very recently, there has been no directtreatment to reduce cerebral tissue damage in evolving stroke. Althoughthe NINDS ² and ECASS³ rt-PA acute stroke studies have suggested thatthere are potential therapeutic benefits of early reperfusion⁴, theincreased mortality observed following streptokinase treatment of acuteischemic stroke⁵ highlights the sobering fact that there is at thepresent time no clearly effective treatment for evolving stroke. Thisvoid in the current medical armamentarium for the treatment of strokehas led to a number of innovative approaches⁶, yet other than rt-PA,none have reached the clinical realm. To identify a potential safe andefficacious treatment for evolving stroke, attention has been focussedon the deleterious role of recruited neutrophils. Recent work in amurine model of reperfused stroke has demonstrated that depletion ofneutrophils (PMNs) prior to stroke minimizes cerebral tissue injury andimproves functional outcome⁷; mice which lack the specific cell adhesionmolecule, ICAM-1, are similarly protected⁷. P-selectin, a molecule whichcan be rapidly translocated to the hypoxic endothelial surface frompreformed storage sites⁸, is an important early mediator of theneutrophil rolling⁹, which facilitates ICAM-1-mediated neutrophilarrest. Although P-selectin is expressed in primate stroke¹⁰, there areno published data which addresses the functional significance ofP-selectin expression in any model of either reperfused or nonreperfusedstroke.

To explore the pathophysiological role of P-selectin in stroke, a murinemodel of focal cerebral ischemia and reperfusion¹¹ was employed usingboth will type mice and mice which were homozygous null for theP-selectin gene⁹ and a strategy of administering a functionally blockingP-selectin antibody. This study confirms not only that P-selectinexpression following middle cerebral artery occlusion is associated withreduced cerebral reflow following reperfusion and a worse outcomefollowing stroke, but that P-selectin blockade confers a significantdegree of postischemic cerebral protection. These studies represent thefirst demonstration of the pathophysiological role of P-selectinexpression in stroke, and suggest the exciting possibility thatanti-P-selectin strategies may prove useful for the treatment ofreperfused stroke.

Methods

Mice

Experiments were performed with transgenic P-selectin deficient mice,created as previously reported⁹ by gene targeting in J1 embryonic stemcells, injected into C57BL/6 blastocysts to obtain germlinetransmission, and backcrossed to obtain homozygous null P-selectin mice(PS −/−). Experiments were performed with PS −/− or wild-type (PS +/+)cousin mice from the third generation of backcrossings with C57BL/6Jmice. Animals were seven to twelve weeks of age and weighed between25-36 grams at the time of experiments.

Transient Middle Cerebral Artery Occlusion

Mice were anesthetized (0.3 cc of 10 mg/cc ketamine and 0.5 mg/ccxylazine, i.p.), and positioned supine on a rectaltemperature-controlled operating surface (Yellow Springs Instruments,Inc., Yellow Springs, Ohio). Animal core temperature was maintained at37+1° C. intraoperatively and for 90 minutes post-operatively. A midlineneck incision was created to expose the right carotid sheath under theoperating microscope (16-25 X zoom, Zeiss, Thornwood, N.Y.). The commoncarotid artery was isolated with a 4-0 silk and the occipital,pterygopalatine, and external carotid arteries were each isolated anddivided. Middle cerebral artery occlusion (MCAO) was accomplished byadvancing a 13 mm heat-blunted 5-0 nylon suture via the external carotidstump. After placement of the occluding suture, the external carotidartery stump was cauterized, and the wound was closed. After 45 minutes,the occluding suture was withdrawn to establish reperfusion. Theseprocedures have been previously described in detail⁹.

Measurement of Cerebral Cortical Blood Flow

Transcranial measurements of cerebral blood flow were made using laserdoppler (Perimed, Inc., Piscataway, N.J.), as previously described¹².Using a 0.7 mm straight laser doppler probe (model #PF303, Perimed,Piscataway, N.J.) and previously published landmarks (2 mm posterior tothe bregma, 6 mm to each side of midline) ^(11,13), relative cerebralblood flow measurements were made as indicated; immediately afteranesthesia, 1 and 10 minutes after occlusion of the middle cerebralartery, as well as after 30 minutes, 300 minutes and 22 hours ofreperfusion. Data are expressed as the ratio of the doppler signalintensity of the ischemic compared with the nonischemic hemisphere.Although this method does not quantify cerebral blood flow per gram oftissue, use of laser doppler flow measurements at precisely definedanatomic landmarks serves as a means of comparing cerebral blood flowsin the same animal serially over time. The surgical procedure wasconsidered to be technically adequate if ≧50% reduction in relativecerebral blood flow was observed immediately following placement of theintraluminal occluding suture. These methods have been used in previousstudies^(7,11).

Cerebrovascular anatomy was determined in representative animals in thefollowing manner. Mice were anesthetized, and an antemortem injection(0.1 mL) of India ink:carbon black:methanol:physiological saline(1:1:1:1, v:v:v:v) was given by left ventricular puncture. Brains wereprepared by rapid decapitation followed by immersion in 10% formalin at4° C. for 2 days, after which the inferior surfaces were photographed todemonstrate the vascular pattern of the Circle of Willis.

Preparation and administration of ¹²⁵ I-labelled proteins and ¹¹¹In-labelled murine neutrophils

Radioiodinated antibodies were prepared as follows. Monoclonal ratanti-murine P-selectin IgG (Clone RB 40.34, Pharmingen Colo., San Diego,Calif.) ¹⁴ and non-immune rat IgG (Sigma Chemical Co., St. Louis, Mo.)were radiolabeled with ¹²⁵I by the lactoperoxidase method ¹⁵ usingEnzymobeads (Bio-Rad, Hercules, Calif.). Radiolabelled PMNs wereprepared in the following manner. Citrated blood from wild type mice wasdiluted 1:1 with NaCl (0.9%) followed by gradient ultracentrifugation onFicoll-Hypaque (Pharmacia, Piscataway, N.J.). Following hypotonic lysisof residual erythrocytes (20 sec exposure to distilled H₂O followed byreconstitution with 1.8% NaCl), the PMNs were suspended in phosphatebuffered saline (PBS). Neutrophils (5-7.5×10⁶) were suspended in PBSwith 100 μCi ¹¹¹ of Indium oxine (Amersham Mediphysics, Port Washington,N.Y.), and subjected to gentle agitation for 15 minutes at 37° C. Afterwashing with PBS, the PMNs were gently pelleted (450×g), and resuspendedin PBS to a final concentration of 1.0×10⁶ cells/mL.

Neurological Exam

Prior to giving anesthesia mice were examined for neurological deficit22 h after reperfusion using a four-tiered grading system¹¹: a score of¹was given if the animal demonstrated normal spontaneous movements; ascore of² was given if the animal was noted to be turning towards theipsilateral side; a score of³ was given if the animal was observed tospin longitudinally (clockwise when viewed from the tail); a score of ⁴was given if the animal was unresponsive to noxious stimuli. Thisscoring system has been previously described in mice^(7,11), and isbased upon similar scoring systems used in rats^(16,17).

Calculation of Infarct Volumes

After neurologic examination, mice were anesthesized and final cerebralblood flow measurements obtained. Humane euthanasia was performed bydecapitation, and brains were removed and place in a mouse brain matrix(Activational Systems Inc., Warren, Mich.) for 1 mm sectioning. Sectionswere immersed in 2% 2,3,5- triphenyl-2H-tetrazolium chloride (TTC, SigmaChemical Co., St. Louis, Mo.) in 0.9% phosphate-buffered saline,incubated for 30 minutes at 37° C., and placed in 10% formalin¹⁸.Infarcted brain was visualized as an area of unstained tissue. Infarctvolumes were calculated from planimetered serial sections and expressedas the percentage of infarct in the ipsilateral hemisphere. This methodof calculating infarct volumes has been used previously^(7,11,13,18),and has been correlated with the other functional indices of strokeoutcome which are described above.

Administration of Unlabeled Antibodies, Radiolabelled PMNs, andRadiolabeled Antibodies

For experiments in which unlabeled antibodies were administered, one oftwo different antibody types was used, either a blocking monoclonal ratanti-murine P-selectin IgG (Clone RB 40.34, Pharmingen Co., San Diego,Calif.)^(14,19,20) or non-immune rat IgG (Sigma Chemical Co., St. Louis,Mo.). Antibodies were prepared as 30 μg in 0.2 mL phosphate bufferedsaline containing 0.1% bovine serum albumin, which was then administeredinto the penile vein 10 minutes prior to middle cerebral arteryocclusion. In separate experiments, radiolabeled antibodies (0.15 mL,≈2.6×10⁵ cpm/μL) were injected intravenously 10 minutes prior to middlecerebral artery occlusion. In a third set of experiments, radiolabelledPMNs were administered intravenously 10 minutes prior to middle cerebralartery occlusion as a 100 μL injection (radiolabelled PMNs were admixedwith physiologic saline to a total volume of 0.15 mL;≈3×10⁶ cpm/μL). Forexperiments in which unlabeled antibodies were administered, the time atwhich measurements were made are indicated in the text, using themethods described above to determine cerebral blood flow, infarctionvolumes, and mortality. For those experiments in which eitherradiolabelled antibodies or radiolabelled nPMNs were administered, micewere sacrificed at the indicated time points and brains were immediatelyremoved and divided into ipsilateral (postischemic) and contralateralhemispheres. Deposition of radiolabeled antibodies or neutrophils wasmeasured and expressed as ipsilateral/contralateral cpm.

Data Analysis

Cerebral blood flow, infarct volume, and ¹¹¹InPMN deposition werecompared using Student's t-test for unpaired variables. Neurologicaldeficit scores were compared using the Mann-Whitney U-test. Two wayANOVA was performed to test for significant differences between baselineand final (30 min) antibody deposition between the two groups(experimental vs sham). Student's t-test for unpaired variables wasperformed to evaluate within-group difference (baseline vs the 30 min.time point). Survival differences between groups was tested usingcontingency analysis with the Chi-square statistic. Values are expressedas mean +SEM, with a p value <0.05 considered statistically significant.

Results

P-selectin Expression in Murine Stroke

Because P-selectin mediates the initial phase of leukocyte adhesion toactivated endothelial cells ²¹, early cerebral P-selectin expression wasexamined in a murine model of reperfused stroke. Mice given a¹²⁵I-labelled rat monoclonal anti-murine P-selectin IgG prior to surgerydemonstrated a 216% increase in accumulation of the antibody at 30minutes of reperfusion compared with sham operated animals (p<0.001,FIG. 18A). To demonstrate that this degree of antibody deposition in thereperfused hemisphere was due to P-selectin expression rather thannonspecific accumulation, comparison was made with identically-treatedanimals given a ¹²⁵I-labelled rat nonimmune IgG. These experimentsdemonstrated that there was significantly greater accumulation of theanti-P-selectin IgG than the nonimmune IgG (p<0.025, FIG. 18A),suggesting that P-selectin is expressed in the brain within 30 minutesof reperfusion.

neutrophil Accumulation in Murine Stroke

To delineate the time course over which PMN influx occurs followingstroke, ¹¹¹In-labeled PMN accumulation was measured in wild type (PS+/+) mice prior to MCAO, immediately following and 10 minutes afterMCAO, and at 30 min, 300 min, and 22 hrs of reperfusion. In PS +/+ mice,accumulation of PMNs begins early following the initiation of focalischemia, and continues throughout the period of reperfusion (FIG. 18B).To establish the role for P-selectin in this postischemic neutrophilaccumulation, experiments were performed using mice which werehomozygous null for the P-selectin gene (PS −/−). PS −/− mice showedsignificantly reduced PMN accumulation following middle cerebral arteryocclusion and reperfusion (FIG. 18B).

Role of PS in Cerebrovascular No-reflow

To determine whether the reduction in PMN accumulation in PS −/− miceresulted in improved cerebral blood flow following the reestablishmentof flow, serial measurements of relative CBF were obtained by laserdoppler in both PS +/+ and PS −/− mice. Prior to the initiation ofischemia (FIG. 19, point a), relative cerebral blood flows were nearlyidentical between groups. Middle cerebral artery occlusion (FIG. 19,point b) was associated with a nearly identical drop in cerebral bloodflow in both groups. Immediately prior to withdrawal of the intraluminaloccluding suture at 45 minutes of ischemia (FIG. 19, point c), cerebralblood flows had risen slightly, although they remained significantlydepressed compared with baseline flows. Immediately following withdrawalof the occluding suture to initiate reperfusion (FIG. 19, point d),cerebral blood flows in both groups increased to a comparable degree(≈60% of baseline in the PS −/− and PS +/+ mice). The immediate failureof the post-reperfusion cerebral blood flows to reach pre-occlusionlevels is characteristic of cerebrovascular no-reflow²², with thesubsequent decline in post-reperfusion cerebral blood flows representingdelayed post-ischemic cerebral hypoperfusion²³. By 30 minutes ofreperfusion (FIG. 19, point e), the cerebral blood flows between the twogroups of animals had diverged, with PS −/− animals demonstratingsignificantly greater relative cerebral blood flows than the PS +/+controls (p<0.05). (FIG. 19, point f). This divergence reflectedsignificant difference in delayed post-ischemic cerebral hypoperfusion,and persisted for the 22 hour observation period.

Because variations in cerebrovascular anatomy have been reported toresult in differences in susceptibility to experimental stroke in mice²⁴, India ink/carbon black staining was performed to visualize the thevascular pattern of the Circle of Willis in both in both PS −/− and PS+/+ mice. These experiments demonstrated that there were no grossanatomic differences in the vascular pattern of the cerebral circulation(FIG. 20).

Stroke Outcome

The functional significance of P-selectin expression was tested bycomparing indices of stroke outcome in PS −/− mice to those in PS +/+controls. PS −/− mice were significantly protected from the effects offocal cerebral ischemia and reperfusion, based on a 77% reduction ininfarct volume (p<0.01) compared with P-selectin +/+ controls (FIG.21A). This reduction in infarct volume was accompanied by a trendtowards reduced neurologic deficit (p=0.06, FIG. 21B) and increasedsurvival (p<0.05; FIG. 21C) in the PS −/− animals.

Effect of P-selectin Blockade

After having observed the functional role of P-selectin expression instroke using deletionally mutant mice, experiments were performed todetermine whether pharmacological blockade of P-selectin could improvestroke outcome in PS +/+ mice. Using a strategy of administering amonoclonal rat anti-mouse P-selectin blocking antibody (clone RB 40.34,^(14,19,20)) or nonimmune control rat IgG immediately prior to surgery,mice receiving the blocking antibody were observed to have improvedpost-reperfusion cerebral blood flows by thirty minutes (FIG. 22A), aswell as reduced neurological deficits (FIG. 22B), reduced cerebralinfarction volumes (FIG. 22C), and a trend towards reduced mortalitycompared with controls (FIG. 22D).

Discussion

Despite substantial progress in recent years in the primary preventionof stroke ¹, therapeutic options to treat evolving stroke remainextremely limited ⁶. Although the publication of two landmark trialslast fall demonstrating reduced morbidity following treatment ofischemic stroke with rt-PA^(2,3) was thought to usher in a new ear ofthrombolytic therapy in the treatment of stroke ⁴, enthusiasm has beentempered somewhat by the hemorrhagic transformation and increasedmortality noted in patients with ischemic stroke treated withstreptokinase ⁵. These divergent trials make it more critical than everthat new safe therapies be developed to treat evolving stroke. Althoughrestoration of blood flow to postischemic brain affords newopportunities for early therapeutic intervention, reperfusion is adouble-edged sword. Given the cytotoxic potential of neutrophils ²⁵, itis not surprising that neutrophil influx into postischemic brain tissuecan lead to further damage and worsen outcome following experimentalstroke^(7,26-29). Using a murine model of focal cerebral ischemia andreperfusion, an important contributory role for the cell adhesionmolecule ICAM-1 in neutrophil accumulation at 22 hours following strokewas recently identified ⁷. However, augmented cerebrovascularendothelial ICAM-1 expression required de novo transcriptional andtranslational events, which requires time. In contrast, P-selectin, amembrane-spanning glycoprotein which mediates the earliest phases ofneutrophil adhesion, may be mobilized from preformed storage pools to berapidly expressed at the ischemic endothelial cell surface^(8,30). Asthe clinical trials of thrombolytic therapy for stroke demonstrate anarrow time window for potential benefit (within the first several hoursof stroke onset) ^(2,3,5), this suggests that strategies designed tointerfere with the earliest phases of PMN adhesion might be oftheoretical benefit in human stroke. These trials should result ingreater numbers of patients presenting for earlier therapeuticintervention, increasing the need to address the issue of reperfusioninjury in medically revascularized territories. In addition, thesetrials underscore the pressing need to understand the contributions ofindividual adhesion molecules to the pathogenesis of stroke.

Given the considerable body of literature describing the role ofP-selectin in other models of ischemia and reperfusion ^(8,31-34),surprisingly little is known about the role of P-selectin in stroke.Knowledge of the specific role of P-selectin in the cerebral vasculatureis important because adhesion molecule requirements vary betweenvascular beds and conditions under study. For instance, in a model ofintestinal transplantation ³⁵, anti-P-selectin antibodies did not reducereperfusion injury, whereas anti-CD11/ CD18 antibodies did. AlthoughP-selectin blockade was ineffective at reducing PMN adhesion and albuminleakage in a rat mesentaric ischemia and reperfusion model, ICAM-1blockade was effective ³⁶. In a rat hindlimb ischemia/reperfusion model,the selectin requirements for PMN adhesion differed between thepulmonary and crural muscle vascular beds ³³.

The only published study dealing with P-selectin in the ischemic brainis a histopathological description of primate stroke, in whichP-selectin expression was increased in the lenticulostriatemicrovasculature ¹⁰. Furthermore, there is no data which addresses thefunctional significance of this P-selectin expression. The currentstudies were undertaken to study whether P-selectin expressioncontributes to post-ischemic cerebral neutrophil accumulation,no-reflow, and tissue injury in a murine model of reperfused stroke.Using a recently established model of focal cerebral ischemia andreperfusion in mice ¹¹, P-selectin expression was demonstrated byincreased deposition of radiolabelled antiody into the ischemicterritory. In this technique, antibody deposition into the ischemichemisphere was normalized to that in the nonischemic hemisphere in eachanimal, not only to minimize potential variations in injection volume orvolume of distribution, but to enable comparison between animals givendifferent antibodies. Because disruption of the endothelial barrierfunction in the ischemic cortex may augment nonselective antibodydeposition, similar experiments were performed with a control rat IgG.These data show that the antibody which binds to P-selectin is depositedat an accelerated rate compared with the control antibody, suggestingthat local P-selectin expression is augmented in the reperfused tissue.This data in the murine model parallels that reported in a baboon modelof stroke ¹⁰, in which P-selectin expression was increased within 1 hourfollowing the ischemic event.

The role of P-selectin expression in recruiting PMNs to thepost-ischemic zone was demonstrated using a strategy in whichaccumulation of ¹¹¹In-labelled PMNs was measured. Although it waspreviously reported that by 22 hours, PMN accumulation is elevated inthe ischemic hemisphere ⁷, the current time-course data demonstrate thatPMN accumulation begins shortly after the onset of ischemia. Failure toexpress the P-selectin gene was associated with reduced PMNaccumulation, suggesting the participation of P-selectin inpost-ischemic cerebral PMN recruitment. However, the P-selectin nullanimals did demonstrate a modes (albeit less than control) neutrophilaccumulation by 22 hours. This data indicates that P-selectin is not theexclusive effector mechanism responsible for postischemic cerebral PMNrecruitment, and is consistent with the previous data that ICAM-1 alsoparticipates in pot-ischemic PMN adhesion ⁷. Furthermore, this data isnot unlike that in which intra-abdominal instillation of thioglycollatein P-selectin deficient mice caused delayed (but not absent) PMNrecruitment ⁹.

Because of the critical need to identify reasons for failed reperfusion,the current studies examined the role of P-selectin in delayedpostischemic cerebral hypoperfusion ^(22,23), the phenomenon whereinblood flow declines during reperfusion, despite restoration of adequateperfusion pressures. In cardiac models of ischemia, no-reflow worsens astime elapses after reperfusion ³⁷, suggesting an important role forrecruited effector mechanisms, such as progressive microcirculatorythrombosis, vasomotor dysfunction, and PMN recruitment. Both P-selectinand ICAM-1-dependent adherence reactions ³⁸ and PMN capillary plugging³⁹ have been shown in other models to participate in post-ischemicno-reflow. In the brain, PMNs have been implicated in post-ischemiccerebral no reflow ^(40,41), but the role of P-selectin in this processhas not been elucidated. The current study uses a relatively noninvasivetechnique (laser doppler) to obtain serial measurements of relativecerebral blood flow, in order to establish the existence, time course,and P-selectin-dependence of post-ischemic cerebrovascular no-reflow. Inthese experiments, P-selectin null and controls animals were subjectedto virtually identical degrees of ischemia, and instantaneous recoveryof blood flow following removal of the intraluminal occluding suture wasthe same in the two groups. However, cerebral blood flow declined in thetime period following reperfusion in P-selectin +/+ animals. In sharpcontrast, the PS −/− animals demonstrated only slight delayedpost-ischemic cerebral hypoperfusion. This late (albeit limited) declinein cerebral blood flow by 22 hours is consistent with the modest PMNrecruitment observed in the PS −/− animals over the same period. Thisagain suggests that other effector mechanisms (such as ICAM-1) may beresponsible for the late decline in cerebral blood flow in PS −/−animals.

The functional effects of P-selectin expression are clear from thecurrent set of studies: animals which fail to express the P-selectingene (or PS +/+ animals treated with a functionally blockinganti-P-selectin antibody) exhibit smaller infarcts, improved survival,and survivors demonstrate improved neurologic outcomes compared withcontrols. When these data are considered along with previously publisheddata demonstrating a deleterious role for ICAM-1 expression in stroke ⁷,it becomes increasingly apparent that there are multiple means forrecruiting PMNs to post-ischemic cerebral cortex, and that blockade ofeach represents a potential strategy to improve stroke outcome inhumans. Given the current recognition of the importance of timelyreperfusion in halting the advancing wavefront of neuronal deathfollowing stroke, interfering with PMN adhesion at its earliest stagesappears to be an attractive option for reducing morbidity and mortality.In fact, anti-adhesion molecule strategies may not only be beneficial intheir own right (i.e., including patients ineligible for thrombolysis),but may extend the window of opportunity for thrombolytic intervention⁴². The current set of studies contributes to the understanding ofpathophysiological mechanisms operative in reperfused stroke. Thesestudies suggest the need for clinical trials of therapies for evolvingstroke which optimize the reperfusion milieu to reduce PMN accumulation.

References:

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13. Huang Z, Huang P L, Panahian N, Dalkara T, Fishman M C, Moskowitz MA: Effects of cerebral ischemia in mice deficient in neuronal nitricoxide synthase. Science 1994;265:1883-1885

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15. David G S, Reisfeld R A: Protein iodination with solid statelactoperoxidase. Biochem 1974;13:1014-1021

16. Bederson J B, Pitts L H, Tsuji M: Rat middle cerebral arteryocclusion: evaluation of the model and development of a neurologicexamination. Stroke 1986;17:472-476

17. Menzies S A, Hoff J T, Betz A L: Middle cerebral artery occlusion inrats: a neurological and pathological evaluation of a reproduciblemodel. Neurosurg 1992;31:100-107

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19. Bosse R, Vestweber D: Only simultaneous blocking of the L- andP-selectin completely inhibits neutrophil migration into mouseperitoneum. Eur J Immunol 1994;24:3019-3024

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21. Springer T A: Adhesion receptors of the immune system. Nature1990;346:425-434

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24. Barone F C, Knudsen D J, Nelson A H, Feuerstein G Z, Willette R N:Mouse strain differences in susceptibility to cerebral ischemia arerelated to cerebral vascular anatomy. J Cereb Blood Flow Metab1993;13:683-692

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26. Hallenbeck J M, Dutka A J, Tanishima T, Kochanek P M, Kumaroo K K,Thompson C B, Obrenovitch T P, Contreras T J: Polymorphonuclearleukocyte accumulation in brain regions with low blood flow during theearly postischemic period. Stroke 1986;17:246-253

27. Kochanek P M, Hallenbeck J M: Polymorphonuclear leukocytes andmonocytes/macrophages in the pathogenesis of cerebral ischemia andstroke. Stroke 1992;23(9):1367-1379

28. Dutka A J, Kochanek P M, Hallenbeck J M: Influence ofgranulocytopenia on canine cerebral ischemia induced by air embolism.Stroke 1989;20:390-395

29. Bednar M M, Raymond S, McAuliffe T, Lodge P A, Gross C E: The roleof neutrophils and platelets in a rabbit model of thromboembolic stroke.Stroke 1991;22(1):44-50

30. Geng J-G, Bevilacqua M P, Moore K L, McIntyre T M, Prescott S M, KimJ M, Bliss G A, Zimmerman G A, McEver R P: Rapid neutrophil adhesion toactivated endothelium mediated by GMP-140. Nature 1990;343:757-760

31. Weyrich A S, Ma X-L, Lefer D J, Albertine K H, Lefer A M: In vivoneutralization of P-selectin protects feline heart and endothelium inmyocardial ischemia and reperfusion injury. J Clin Invest1993;91:2620-2629

32. Winn R K, Liggitt D, Vedder N B, Paulson J C, Harlan J M:Anti-P-selectin monoclonal antibody attenuates reperfusion injury in therabbit ear. J Clin Invest 1993;92:2042-2047

33. Seekamp A, Till G O, Mulligan M S, Paulson J C, Anderson D C,Miyasaka M, Ward P A: Role of selectins in local and remote tissueinjury following ischemia and reperfusion. Am J Pathol 1994;144:592-598

34. Kubes P, Jutila M, Payne D: Therapeutic potential of inhibitingleukocyte rolling in ischemia/reperfusion. J Clin Invest1995;95:2510-2519

35. Slocum M M, Granger D N: Early mucosal and microvascular changes infeline intestinal transplants. Gastroenterology 1993;105:1761-1768

36. Kurose I, Anderson D C, Miyasaka M, Tamatani T, Paulson J C, Todd RF, Rusche J R, Granger D N: Molecular determinants ofreperfusion-induced leukocyte adhesion and vascular protein leakage.Circ Res 1994;74:336-343

37. Kloner R A, Ganote C E, Jennings R B: The “no-reflow” phenomenonafter temporary coronary occlusion in the dog. J Clin Invest1974;54:1496-1508

38. Jerome S N, Dore M, Paulson J C, Smith C W, Korthius R J: P-selectinand ICAM-1-dependent adherence reactions: role in the genesis ofpostischemic no-reflow. Am J Physiol 1994;266:H1316-H1321

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40. Mori E, del Zoppo G J, Chambers J D, Copeland B R, Arfors K E:Inhibition of polymorphonuclear leukocyte adherence suppresses no-reflowafter focal cerebral ischemia in baboons. Stroke 1992;23:712-718

41. Grogaard B, Schurer L, Gerdin B, Arfors K E: Delayed hypoperfusionafter incomplete forebrain ischemia in the rat: the role ofpolymorphonuclear leukocytes. J Cereb Blood Flow Metab 1989;9:500-505

42. Bowes M P, Rothlein R, Fagan S C, Zivin J A: Monoclonal antibodiespreventing leukocyte activation, reduce experimental neurologicalinjury, and enhance efficacy of thrombolytic therapy. Neurology1995;45:815-819

EXAMPLE 5 P-Selectin Homozygous Null Mice Are Resistant to FocalCerebral Ischemia and Reperfusion Injury

The role of neutrophils (PMNs) in potentiating focal ischemiareperfusion injury in the central nervous system remains controversial.An important early step in the capture of circulating PMNs by thevasculature is mediated by P-selectin expressed on postischemicendothelium. Although early and persistent endothelial P-selectinexpression has been described in brain microvessels following middlecerebral artery occlusion in baboons, the consequences of endothelialP-selection expression in stroke have not been determined. To define therole of P-selectin in stroke, a murine model of focal cerebral ischemiaand reperfusion consisting of intraluminal middle cerebral artery (MCA)occlusion for 45 minutes followed by 22 hours of reperfusion was used intwo groups of mice; transgenic mice that were homozygous null forP-selectin (PS −/−), and wild type cousin controls (PS +/+). Cerebralinfarct volumes were calculated from planimetered serial sectionsstained with triphenyltetrazolium chloride, and expressed as thepercentage of infarcted tissue in the ipsilateral hemisphere. Neurologicoutcome was based on animal behavior observed by a blinded investigator(1: no deficit; 2: circling; 3: spinning; 4: immobile). Ipsilateralcortical cerebral blood flow (CBF) was determined by laser dopplerflowmetry and expressed as percent of contralateral cortical CBF. PS −/−mice showed a 3.8-fold reduction in infarct volumes compared with PS +/+controls (7.6±4.4% vs 29.2±10.1%, p<0.05). This reduction in infarctvolumes in mice devoid of P-selectin was mirrored by improved survival(87% vs. 42%, p<0.05) and a trend towards reduced neurological deficit(1.9±0.4 vs. 2.5±0.3, p=NS) in survivors. Because there was a tendencyfor increased cerebral blood flow following cerebral ischemia andreperfusion in the PS −/− cohort (65±11% vs. 46±18% for controls, p=NS),these studies suggest that P-selectin-dependent adhesion may contributeto cerebral no-reflow. Taken together, these data implicate an importantrole for P-selectin expression in the pathophysiology of stroke, andsuggest novel pharmacologic strategies to improve stroke outcome.

EXAMPLE 6 Absence of the P-selectin Gene Reduces Post-ischemic CerebralNeutrophil Accumulation, No-reflow, and Tissue Injury in a Murine Modelof Reperfused Stroke

Recent studies in humans indicate that reestablishment of cerebral bloodflow (CBF) during the early period following the onset of stroke reducesneurologic sequelae. It was hypothesized that P-selectin (PS), anearly-acting neutrophil (PMN) adhesion molecule expressed by hypoxicendothelium may have an important pathophysiological role in evolving,reperfused stroke. Preliminary studies were performed in a murine modelof transient focal cerebral ischemia consisting of intraluminal middlecerebral artery occlusion of 45 minutes followed by 22 hours ofreperfusion. In this model, mice which do not express the PS gene (PS−/−) have a smaller infarct volumes, reduced neurological deficitscores, and improved survival compared to wild-type controls (PS +/+).The current studies were performed to further define PS-inducedmechanism(s) of cerebral injury. PS +/+ mice (N=6) given a ¹²⁵I-labeledanti-PS IgG prior to surgery demonstrated a 216% greater accumulation ofthe antibody in the ipsilateral hemisphere by 30 min of reperfusioncompared with sham-operated animals (n=6, p<0.001) or with animals givennonimmune IgG and subjected to transient focal cerebral ischemia and 30min of reperfusion (n=4, p<0.03). In PS +/+ mice, accumulation of PMNsbegins early following the initiation of focal ischemia, and continuesthroughout the period of reperfusion (2-fold increase inipsilateral/contralateral ¹¹¹In-PMN accumulation by 22 hours, n=8,p<0.05). PS −/− mice showed a 25% reduction in PMN accumulation into theipsilateral hemisphere by 22 hours (n=7, p<0.05). The effect of PSexpression on post-ischemic cerebral no-reflow was investigated bymeasuring ipsilateral CBF serially during stroke evolution. Althoughbaseline, post-occlusion, and initial reperfusion CBFs were identical,CBFs at 30 minutes of reperfusion were significantly greater in PS −/−mice (n=5) compared to PS +/+ mice (n=8, 2.4-fold greater, p<0.05). Thisdifference was sustained during the remainder of the 22 h reperfusionperiod. These data support an important early role for PS in PMNrecruitment, post-ischemic no-reflow, and tissue damage in evolvingstroke. This is the first demonstration of a pathophysiological role forPS in cerebral reperfusion injury, which suggests that PS blockade mayrepresent a therapeutic target for the treatment of reperfused stroke.

EXAMPLE 7 Carbon Monoxide and Evolving Stroke

Carbon monoxide gas, a toxic byproduct of heme catabolism, is involvedin long-term potentiation and memory in the central nervous system.However, other physiologic roles for CO production in the brain areunknown. Because heme oxygenase is induced during inflammatoryconditions, it was investigated whether endogenous CO production mayconfer a cerebral protective role in stroke. In a murine model of focalcerebral ischemia, heme oxygenase type I was induced at the mRNA (byNorthern blot) and protein levels (by Western blot), localized to thecerebral vascular endothelium in the ischemic hemisphere by in situhybridization and immunohistochemistry. Local production of CO by directmeasurement was observed in the ischemic zone. In parallel experiments,murine brain endothelial cells exposed to a hypoxic environmentdemonstrated similar induction of heme oxygenase mRNA, protein and COgeneration. To determine whether CO production was incidental to thepathophysiology of stroke, CO production was blocked by tinprotoporphyrin administration (confirmed by direct measurement ofreduced local CO levels). These animals demonstrated significantlylarger infarct volumes, worse neurological outcomes, and increasedmortality compared with untreated controls. Furthermore, administrationof CO prior to stroke conferred significant cerebral protection. As thisprotection was not observed in animals treated with biliverdin, thecoincident byproduct of heme catabolism, these data suggest thatendogenous CO production per se has a protective role in evolvingstroke.

Introduction

There is a considerable body of literature and a common recognition ofthe toxic effects of exogenous carbon monoxide (CO), which binds avidlyto heme centers, inhibiting oxygen transport and poisoning cellularrespiration. For many years, CO was regarded as an incidental byproductof heme catabolism, but recent data in the brain suggests that COproduced in discrete neurons by heme oxygenase II may modulate long-termpotentiation. In rats, exposure to heat shock has been correlated withthe expression of a 32 kDa heat shock protein (heme oxygenase I) inseveral organs including the brain. The physiological significance ofthis HSP32 induction has been teleologically ascribed to thestoichiometric liberation of CO by heme oxygenase I (HOI). In most,experimental studies, HOI induction serves only as an incidental markerof cellular oxidant stress. The recent identification of ananti-inflammatory role for HOI in a model of peritoneal inflammation hasbeen ascribed to the production of the natural antioxidant biliverdinduring the process of heme catabolism.

The current study reports for the first time that the postischemic braingenerates enormous quantities of CO. Using a murine model of focalcerebral ischemia in which the middle cerebral artery is occluded by anintraluminal suture, HOI production in the ischemic hemisphere wasincreased significantly in comparison to the nonischemic hemisphere.Because immunohistochemistry and in situ hybridization localized thesource of HOI to endothelial cells within the ischemic hemisphere, an invitro model of cellular hypoxia was used to confirm the induction of HOImessage, protein and activity in murine cerebral microvascularendothelial cells. Blockade of CO production using tin or zincprotoporphyrin IX was associated with an increase in cerebral infarctvolume and mortality, whereas exposing animals to CO immediately priorto ischemia conferred significant dose-dependent cerebral protectionwithin a narrow therapeutic window. Biliverdin administration waswithout effect in this model. Taken together, these data indicate thatischemic brain tissue produces large amounts of CO, the production ofwhich confers cerebral protection that limits the amount of tissuedestroyed during stroke.

Methods

Protoporphyrin preparation and administration. Tin protoporphyrin IXdichloride (20 mg, Porphyrin Products, Logan, Utah), zinc protoporphyrinIX (17 mg, Porphyrin Products, Logan, Utah), or biliverdin (18 mg,Porphyrin Products, Logan, Utah) was initially dissolved in dimethylsulfoxide (2 mL). An aliquot of this solution (200 μL) was added tonormal saline (9.8 mL) and this mixture was vortexed vigorously to yielda 2.7×10⁻⁴ M solution of the protoporphyrin. The solution container waswrapped in aluminum foil to prevent photolysis of the protoporphyrin andstored at 4° C. until used.

Micro-osmotic pumps (#1003D, Alza Corp., Palo Alto, Calif.) were loadedwith this protoporphyrin solution (91 μL/pump) and implantedsubcutaneously in the anesthetized mouse via a 1 cm dorsal midlineincision 24 h prior to the start of surgery. These pumps administer drugsolution at a rate of 0.95±0.02 μL/h. At the time of surgery anadditional dose of the protoporphyrin solution was administered (0.3 mL,i.v.) prior to insertion of the intralumenal occluding catheter. Eachanimal received the following total (injection+pump) drug amounts overthe course of the study: tin protoporphyrin (0.070 mg), zincprotoporphyrin (0.059 mg), or biliverdin (0.061 mg).

EXAMPLE 8

Hypoxia or free radicals induce P-selectin (PS) translocation to theendothelial cell (EC) surface, where it participates in neutrophil (PMN)adhesion during reperfusion. To explore a mechanism wherebynitrovasodilators may attenuate postischemic leukosequestration, wetested whether stimulating the NO/cGMP pathway could attenuate surfacePS expression in hypoxic human umbillical vein ECs. ECs exposed tohypoxia (pO2<20 Torr for 4 hours) demonstrated a 50% increase in νWFrelease (p<0.005) (νWF is packaged with PS), paralleled by an 80%increase in surface PS expression (p<0.0001), measured by specificbinding of a radiolabeled anti-PS antibody. Under similar conditions,addition of the NO donor 3-morpholino sydnonimine (SIN-1, 0.1 mM) or thecGMP analog 8-Bromo-cGMP (cGMP, 10 nM) caused a reduction in νWFrelease; Control νWF, 11±0.4 mU/mL; SIN-1, 9.1±0.3 mU/mL; cGMP, 9.7±0.2mU/mL; p<0.005 for both SIN-1 or cGMP vs Control. Compared withcontrols, SIN-1 or cGMP also reduced surface PS expression (40% and 48%decreases respectively, p<0.005 for each) Using an immunofluorescentadherence assay, both SIN-1 and cGMP reduced HL60 binding to hypoxicHUVECs (53% and 86% decrease vs. controls, p<0.05 for each). Measurementof fura-2 fluorescence demonstrated that hypoxia increased intracellularcalcium concentration [Cai], and that increased [Cai] could be blockedby cGMP. Neither SIN-1 nor cGMP could further reduce PS expression whenECs were placed in a calcium-free medium. These data suggest thatstimulation of the NO/cGMP pathway inhibits PS expression by inhibitingcalcium-flux in ECs, and identify this inhibition as an importantmechanism whereby nitrovasodilators may decrease PMN binding inpost-ischemic tissues.

EXAMPLE 9 Factor IXai

Factor IX is a clotting factor which exists in humans and other mammals,and is an important part of the coagulation pathway. In the normalscheme of coagulation, Factor IX is activated by either Factor XIa or atissue factor/VIIa complex to its active form, Factor IXa. Factor IXathen can activate Factor X, which triggers the final part of thecoagulation cascade, leading to thrombosis. Because Factor X can beactivated by one of two pathways, either the extrinsic (via VIIa/tissuefactor) or the intrinsic pathways (via Factor IXa), we hypothesized thatinhibiting Factor IXa might lead to impairment of some forms ofhemostasis, but lease hemostasis in response to tissue injury intact. Inother words, it might lead to blockade of some types of clotting, butmight not lead to excessive or unwanted hemorrhage. Factor IXai isFactor IX which has been chemically modified so as to still resembleFactor IXa (ant therefore, can compete with native Factor IXa), butwhich lacks its activity. This can “overwhelm” or cause a competitiveinhibition of the normal Factor IXa-dependent pathway of coagulation.Because Factor IXa binds to endothelium and platelets and perhaps othersites, blocking the activity of Factor IXa may also be possible byadministering agents which interfere with the binding of Factor IXa (orby interfering with the activation of Factor IX).

In stroke and other ischemic disorders, there may be clinical benefitderived by lysing an existing thrombus, but there is also thepotentially devastating complication of hemorrhage. In the currentexperiments, the mouse model of cerebral ischemia and reperfusion(stroke) was used. Mice received an intravenous bolus of 300 μg/kg ofFactor IXai just prior to surgery. Strokes were created by intraluminalocclusion of the right middle cerebral artery. When stroke outcomes weremeasured 24 hours later, animals that had received Factor IXai hadsmaller infarct volumes, improved cerebral perfusion, less neurologicaldeficits, and reduced mortality compared with controls which underwentthe same surgery but which did not receive Factor IXai. It was alsonoted that the Factor IXai animals were free of apparent intracerebralhemorrhage. By contrast, intracerebral hemorrhage was occasionally notedin the control animals not receiving Factor IXai.

TABLE II Control Experimental mean sd mean sd stats weight 26.91 3.2125.25 2.49 0.14 dopp 0.96 0.24 1.04 0.35 0.52 occ dop 1 0.18 0.07 0.160.08 0.60 occ dop 2 0.40 0.22 0.43 0.20 0.68 reper dop 0.55 0.42 0.530.30 0.89 sac dop 0.38 0.25 0.75 0.31 0.02 grade 2.22 0.67 1.67 0.49 I/CRatio 1.18 0.20 1.08 inf vol 21.16 25.14 3.47 12.03 0.0452 count 11 16

EXAMPLE 10 Exacerbation of Cerebral Injury In Mice Which Express theP-Selectin Gene: Identification of P-selectin Blockade as a New Targetfor the Treatment of Stroke

Abstract:

There is currently a stark therapeutic void for the treatment ofevolving stroke. Although P-selectin is rapidly expressed by hypoxicendothelial cells in vitro, the functional significance of P-selectinexpression in stroke remains unexplored. In order to identify thepathophysiological consequences of P-selectin expression and to identifyP-selectin blockade as a potential new approach for the treatment ofstroke, experiments were performed using a murine model of focalcerebral ischemia and reperfusion. Early P-selectin expression in thepost-ischemic cerebral cortex was demonstrated by the specificaccumulation of radiolabelled anti-murine P-selectin IgG, with theincreased P-selectin expression localized to the ipsilateral cerebralmicrovascular endothelial cells by immunohistochemistry. In experimentsdesigned to test the functional significance of increased P-selectinexpression in stroke, neutrophil accumulation in the ischemic cortex ofmice expressing the P-selectin gene (PS +/+) was demonstrated to besignificantly greater than that in homozygous P-selectin null mice (PS−/−). Reduced neutrophil influx was accompanied by greater postischemiccerebral reflow (measured by laser doppler) in the PS −/− mice. Inaddition, PS −/− mice demonstrated smaller infarct volumes (five-foldreduction, p<0.05) and improved survival compared with PS +/+ mice (88%vs. 44%, p<0.05). Functional blockade of P-selectin in PS +/+ mice usinga monoclonal antibody directed against murine P-selectin also improvedearly reflow and stroke outcome compared with controls, with reducedcerebral infarction volumes noted even when the blocking antibody wasadministered after occlusion of the middle cerebral artery. These dataare the first to demonstrate a pathophysiological role for P-selectin instroke, and suggest that P-selectin blockade may represent a newtherapeutic target for the treatment of stroke.

Introduction:

Ischemic stroke constitutes the third leading cause of death in theUnited States today¹. Until very recently, there has been no directtreatment to reduce cerebral tissue damage in evolving stroke. Althoughthe NINDS² and ECAS³S rt-PA^(2†) acute stroke studies have suggestedthat there are potential therapeutic benefits of early reperfusion⁴, theincreased mortality observed following streptokinase treatment of acuteischemic stroke⁵ highlights the sobering fact that there is at thepresent time no clearly effective treatment for evolving stroke. Thisvoid in the current medical armamentarium for the treatment of strokehas led to a number of innovative approaches⁶, yet other than rt-PA,none have reached the clinical realm. To identify a potential safe andefficacious treatment for evolving stroke, we have focussed on thedeleterious role of recruited neutrophils. Recent work in a murine modelof reperfused stroke has demonstrated that depletion of neutrophils(PMNs) prior to stroke minimizes cerebral tissue injury and improvesfunctional outcome⁷; mice which lack the specific cell adhesionmolecule, ICAM-1, are similarly protected⁷. P-selectin, a molecule whichcan be rapidly translocated to the hypoxic endothelial surface frompre-formed storage sites⁸, is an important early mediator of theneutrophil rolling⁹, which facilitates ICAM-1-mediated neutrophilarrest. Although P-selectin is expressed in primate stroke¹⁰, thefunctional significance of P-selectin expression in stroke remainsunknown.

To explore the pathophysiological role of P-selectin in stroke, weemployed a murine model of focal cerebral ischemia and reperfusion¹¹using both wild type mice and mice which were homozygous null for theP-selectin gene⁹ and a strategy of administering a functionally blockingP-selectin antibody. In these studies, we confirm not only thatP-selectin expression following middle cerebral artery occlusion isassociated with reduced cerebral reflow following reperfusion and aworse outcome following stroke, but that P-selectin blockade confers asignificant degree of postischemic cerebral protection. These studiesrepresent the first demonstration of the pathophysiological role ofP-selectin expression in stroke, and suggest the exciting possibilitythat anti-P-selectin strategies may prove useful for the treatment ofreperfused stroke.

Methods:

Mice: Experiments were performed with transgenic P-selectin deficientmice created as previously reported⁹ by gene targeting in J1 embryonicstem cells, injected into C57BL/6 blastocysts to obtain germlinetransmission, and backcrossed to obtain homozygous null P-selectin mice(PS −/−). Experiments were performed with PS −/− or wild-type (PS +/+)cousin mice from the third generation of backcrossings with C57BL/6Jmice. Animals were seven to twelve weeks of age and weighed between25-36 grams at the time of experiments. Because variations incerebrovascular anatomy have been reported to result in differences insusceptibility to experimental stroke in mice¹², India ink/carbon blackstaining was performed to visualize the the vascular pattern of theCircle of Willis in both in both PS −/− and PS +/+ mice. Theseexperiments demonstrated that there were no gross anatomic differencesin the vascular pattern of the cerebral circulation.

Transient Middle Cerebral Artery Occlusion: Mice were anesthetized (0.3cc of 10 mg/cc ketamine and 0.5 mg/cc xylazine, i.p.), and positionedsupine on a rectal temperature-controlled operating surface (YellowSprings Instruments, Inc., Yellow Springs, Ohio). Animal coretemperature was maintained at 37±1° C. intraoperatively and for 90minutes post-operatively. A midline neck incision was created to exposethe right carotid sheath under the operating microscope (16-25 X zoom,Zeiss, Thornwood, N.Y.). The common carotid artery was isolated with a4-0 silk, and the occipital pterygopalatine, and external carotidarteries were each isolated and divided. Middle cerebral arteryocclusion (MCAO) was accomplished by advancing a 13 mm heat-blunted 5-0nylon suture via the external carotid stump. After placement of theoccluding suture, the external carotid artery stump was cauterized, andthe wound was closed. After 45 minutes, the occluding suture waswithdrawn to establish reperfusion. These procedures have beenpreviously described in detail¹¹.

Measurement of cerebral cortical blood flow: Transcranial measurement ofcerebral blood flow were made using laser doppler (Perimed Inc.,Piscataway, N.J.), and previously described¹³. Using a 0.7 mm straightlaser doppler probe (model #PF303, Perimed, Piscataway, N.J.) andpreviously published landmarks (2 mm posterior to the bregma, 6 mm toeach side of midline)¹¹, relative cerebral blood flow measurements weremade as indicated; immediately after anesthesia, 1 and 10 minutes afterocclusion of the middle cerebral artery, as well as after 30 minutes,300 minutes and 22 hours of reperfusion. Data are expressed as the ratioof the doppler signal intensity of the ischemic compared with thenonischemic hemisphere. Although this method does not quantify cerebralblood flow per gram of tissue, use of laser doppler flow measurements atprecisely defined anatomic landmarks serves as a means of comparingcerebral blood flows in the same animal serially over time. The surgicalprocedure was considered to be technically adequate if ≧50% reduction inrelative cerebral blood flow was observed immediately followingplacement of the intraluminal occluding suture. These methods have beenused in previous studies^(7,11).

Preparation and administration of 125I-labelled proteins and¹¹¹In-labelled murine neutrophils: Radioiodinated antibodies wereprepared as follows. Monoclonal rat anti-murine P-selectin IgG (Clone RB40.34, Pharmingen Co., San Diego, Calif.)¹⁴ and non-immune rat IgG(Sigma Chemical Co., St. Louis, Mo.) were radiolabeled with ¹²⁵I by thelactoperoxidase method¹⁵ using Enzymobeads (Bio-Rad, Hercules, Calif.).Radiolabelled PMNs were prepared in the following manner. Citrated bloodfrom wild type mice was diluted 1:1 with NaCl (0.9%) followed bygradient ultracentrifugation on Ficoll-Hypaque (Pharmacia, Piscataway,N.J.). Following hypotonic lysis of residual erythrocytes (20 secexposure to distilled H₂O followed by reconstitution with 1.8% NaCl),the PMNs were suspended in phosphate buffered saline (PBS). Neutrophils(5-7.5×10⁶) were suspended in PBS with 100 μCi of ¹¹¹ Indium oxine(Amersham Mediphysics, Port Washington, N.Y.), and subjected to gentleagitation for 15 minutes at 37° C. After washing with PBS, the PMNs weregently pelleted (450× g), and resuspended in PBS to a finalconcentration of 1.0×10⁶ cells/mL.

Calculation of Infarct Volumes: After neurologic examination, mice wereanesthesized and final cerebral blood flow measurements obtained. Humaneeuthanasia was performed by decapitation, and brains were removed andplaced in a mouse brain matrix (Activational Systems Inc., Warren,Mich.) for 1 mm sectioning. Sections were immersed in 2%2,3,5-triphenyl-2H-tetrazolium chloride (TTC, Sigma Chemical Co., St.Louis, Mo.) in 0.9% phosphate-buffered saline, incubated for 30 minutesat 37° C., and placed in 10% formalin¹⁶. Infarcted brain was visualizedas an area of unstained tissue. Infarct volumes were calculated fromplanimetered serial sections and expressed as the percentage of infarctin the ipsilateral hemisphere. This method of calculating infarctvolumes has been used previously by our group^(7,11) and others^(16,17),and has been correlated with the other functional indices of strokeoutcome which are described above.

Administration of Unlabelled Antibodies, Ratiolabelled PMNs, andRatiolabelled Antibodies: For experiments in which unlabelled antibodieswere administered, one of two different antibody types was used; eithera blocking monoclonal rat anti-murine P-selectin IgG (Clone RB 40.34,Pharmingen Co., San Diego, Calif.)^(14,18,19) or non-immune rat IgG(Sigma Chemical Co., St. Louis, Mo.). Antibodies were prepared as 30 μgin 0.2 mL phosphate buffered saline containing 0.1% bovine serumalbumin, which was then administered into the penile vein 10 minutesprior to middle cerebral artery occlusion. In separate experiments,radiolabelled antibodies (0.15 mL, ≈2.6×10⁵ cpm/μL) were injectedintravenously 10 minutes prior to middle cerebral artery occlusion. In athird set of experiments, radiolabelled PMNs were administeredintravenously 10 minutes prior to middle cerebral artery occlusion as a100 μL injection (radiolabelled PMNs were admixed with physiologicsaline to a total volume of 0.15 mL; ≈3×10⁶ cpm/μL). For experiments inwhich unlabelled antibodies were administered, the time at whichmeasurements were made are indicated in the text, using the methodsdescribed above to determine cerebral blood flow, infarction volumes,and mortality. For those experiments in which either radiolabelledantibodies or radiolabelled PMNs were administered, mice were sacrificedat the indicated time points and brains were immediately removed anddivided into ipsilateral (postischemic) and contralateral hemispheres.Deposition of radiolabeled antibodies or neutrophils was measured andexpressed as ipsilateral/contralateral cpm.

Immunohistochemistry: Brains were removed at 1 hour following middlecerebral artery occlusion, fixed in 10% formalin, paraffin embedded andsectioned for immunohistochemistry. Sections were stained with anaffinity-purified polyclonal rabbit anti-human P-selectin antibody (1:25dilution, Pharmingen, San Diego, Calif.), and sites of primary antibodybinding were visualized using a biotin-conjugated goat anti-rabbit IgG(1:20) detected with ExtrAvidin peroxidase (Sigma Chemical Co., St.Louis, Mo.).

Data Analysis: Cerebral blood flow, infarct volume, and ¹¹¹In-PMNdeposition were compared using Student's t-test for unpaired variables.Two way ANOVA was performed to test for significant differences betweenbaseline and final (30 min) antibody deposition between the two groups(experimental vs sham). Student's t-test for unpaired variables wasperformed to evaluate within-group differences (baseline vs the 30 min.time point). Survival differences between groups was tested usingcontingency analysis with the Chi-square statistic. Values are expressedas mean±SEM, with a p value<0.05 considered statistically significant.

Results:

P-selectin Expression in Murine Stroke: Because P-selectin mediates theinitial phase of leukocyte adhesion to activated endothelial cells²⁰, weexamined early cerebral P-selectin expression in a murine model ofreperfused stroke. Mice given a ¹²⁵I-labelled rat monoclonal anti-murineP-selectin IgG prior to surgery demonstrated a 216% increase inaccumulation of the antibody at 30 minutes of reperfusion compared withsham operated animals (p<0.001, FIG. 31A). To demonstrate that thisdegree of antibody deposition in the reperfused hemisphere was due toP-selectin expression rather than nonspecific accumulation, comparisonwas made with identically-treated animals given a ¹²⁵I-labelled ratnonimmune IgG. These experiments demonstrated that there wassignificantly greater accumulation of the anti-P-selectin IgG than thenonimmune IgG (p<0.025, FIG. 31A), suggesting that P-selectin isexpressed in the brain within 30 minutes of reperfusion. Examination ofsections of brain tissue immunostained for P-selectin reveal thatP-selectin expression is primarily localized to the microvascularendothelial cells in the ipsilateral cerebral cortex (FIG. 31B).

Neutrophil Accumulation in Murine Stroke: To delineate the time courseover which PMN influx occurs following stroke, ¹¹¹In-labeled PMNaccumulation was measured in wild type (PS +/+) mice prior to MCAO,immediately following and 10 minutes after MCAO, and at 30 min, 300 min,and 22 hrs of reperfusion. In PS +/+ mice, accumulation of PMNs beginsearly following the initiation of focal ischemia, and continuesthroughout the period of reperfusion (FIG. 31C). To establish the rolefor P-selectin in this postischemic neutrophil accumulation, experimentswere performed using mice which were homozygous null for the P-selectingene (PS −/−). PS −/− mice showed significantly reduced PMN accumulationfollowing middle cerebral artery occlusion and reperfusion (FIG. 31B).

Role of P-selectin in Cerebrovascular No-reflow: To determine whetherthe reduction in PMN accumulating in PS −/− mice resulted in improvedcerebral blood flow following the reestablishment of flow, serialmeasurements of relative CBF were obtained by laser doppler in both PS+/+ and PS −/− mice. Prior to the initiation of ischemia (FIG. 32, pointa), relative cerebral blood flows were nearly identical between groups.Middle cerebral artery occlusion (FIG. 32, point b) was associated witha nearly identical drop in cerebral blood flow in both groups.Immediately prior to withdrawal of the intraluminal occluding suture at45 minutes of ischemia (FIG. 32, point c), cerebral blood flows hadrisen slightly, although they remained significantly depressed comparedwith baseline flows. Immediately following withdrawal of the occludingsuture to initiate reperfusion (FIG. 32, point d), cerebral blood flowsin both groups increased to a comparable degree (≈60% of baseline in thePS −/− and PS +/+ mice). The immediate failure of the post-reperfusioncerebral blood flows to reach pre-occlusion levels is characteristic ofcerebrovascular no-reflow²¹, with the subsequent decline inpost-reperfusion cerebral blood flows representing delayed post-ischemiccerebral hypoperfusion²². By 30 minutes of reperfusion (FIG. 32, pointe), the cerebral blood flows between the two groups of animals haddiverged, with PS −/− animals demonstrating significantly greaterrelative cerebral blood flows than the PS +/+ controls (p<0.05). (FIG.32, point f). This divergence reflected significant differences indelayed post-ischemic cerebral hypoperfusion, and persisted for the 22hour observation period.

Stroke Outcome: The functional significance of P-selectin expression wastested by comparing indices of stroke outcome in PS −/− mice to those inPS +/+ controls. PS −/− mice were significantly protected from theeffects of focal cerebral ischemia and reperfusion, based on a 77%reduction in infarct volume (p<0.01) compared with P-selectin +/+controls (FIG. 33A). This reduction in infarct volume was accompanied byincreased survival in the PS −/− animals (p<0.05; FIG. 33B).

Effect of P-selectin Blockade: After having observed the functional roleof P-selectin expression in stroke using deletionally mutant mice,experiments were performed to determine whether pharmacological blockadeof P-selectin could improve stroke outcome in PS +/+ mice. Using astrategy of administering a functionally blocking monoclonal ratanti-mouse P-selectin antibody (clone RB 40.34^(14,18,19)) or nonimmunecontrol rat IgG immediately prior to surgery, mice receiving theblocking antibody immediately prior to middle cerebral artery occlusionwere observed to have improved post-reperfusion cerebral blood flows bythirty minutes, as well as reduced cerebral infarction volumes and atrend towards reduced mortality compared with controls (FIG. 34,leftmost 6 bars). To increase the potential clinical relevance of astrategy of P-selectin blockade as a new treatment for stroke,additional experiments were performed in which either the control or theblocking antibody were given after intraluminal occlusion of the middlecerebral artery (because most patients present following the onset ofstroke). In these studies, a significant reduction in infarct volumeswas observed as well as a trend towards improved cerebral blood flow(FIG. 34, rightmost 6 bars).

Discussion:

Despite substantial progress in recent years in the primary preventionof stroke¹, therepeutic options to treat evolving stroke remainextremely limited. Although the publication of two landmark trials lastfall demonstrating reduced morbidity following treatment of ischemicstroke with rt-PA^(2,3) was thought to usher in a new era ofthrombolytic therapy in the treatment of stroke⁴, enthusiasm has beentempered somewhat by the hemorrhagic transformation and increasedmortality noted in patients with ischemic stroke treated withstreptokinase⁵. These divergent trials make it more critical than everthat new safe therapies be developed to treat evolving stroke. Althoughrestoration of blood flow to postischemic brain affords newopportunities for early therapeutic intervention, reperfusion is adouble-edged sword. Given the cytotoxic potential of neutrophils²³, itis not surprising that neutrophil influx into postischemic brain tissuecan lead to further damage and worsen outcome following experimentalstroke^(7,24-27). Using a murine model of focal cerebral ischemia andreperfusion, we have recently identified an important contributory rolefor the cell adhesion molecule ICAM-1 in neutrophil accumulation at 22hours following stroke⁷. However, augmented cerebrovascular endothelialICAM-1 expression requires de novo transcriptional and translationalevents, which requires time. In contrast, P-selectin, amembrane-spanning glycoprotein which mediates the earliest phase ofneutrophil adhesion, may be mobilized from preformed storage pools to berapidly expressed at the ischemic endothelial cell surface^(8,28). Asthe clinical trials of thrombolytic therapy for stroke demonstrate anarrow time window for potential benefit (within the first several hoursof stroke onset)^(2,3,5), this suggests that strategies designed tointerfere with the earliest phases of PMN adhesion might be oftheoretical benefit in human stroke. These trials should result ingreater numbers of patients presenting for earlier therepeuticintervention, increasing the need to address the issue of reperfusioninjury in medically revascularized territories. In addition, theyunderscore the pressing need to understand the contributions ofindividual adhesion molecules to the pathogenesis of stroke.

Given the considerable body of literature describing the role ofP-selectin in other models of ischemia and reperfusion^(8,29-32),surprisingly little is known about the role of P-selectin in stroke.Knowledge of the specific role of P-selectin in the cerebral vasculatureis important because adhesion molecule requirements vary betweenvascular beds and conditions under study. For instance, in a model ofintestinal transplantation³³, anti-P-selectin antibodies did not reducereperfusion injury, whereas anti-CD11/CD18 antibodies did. AlthoughP-selectin blockade was ineffective at reducing PMN adhesion and albuminleakage in a rat mesenteric ischemia and reperfusion model, ICAM-1blockade was effective³⁴. In a rat hind limb ischemia/reperfusion model,the selectin requirements for PMN adhesion differed between thepulmonary and crural muscle vascular beds³¹.

To our knowledge, the only published study describing increasedP-selectin expression in the ischemic brain is a histopathologicaldescription of primate stroke, in which P-selectin expression wasincreased in the lenticulostriate microvasculature¹⁰. The currentstudies were undertaken to study whether P-selectin expressioncontributes to post-ischemic cerebral neutrophil accumulation,no-reflow, and tissue injury in a murine model of reperfused stroke.Using a recently established model of focal cerebral ischemia andreperfusion in mice¹¹, P-selectin expression was demonstrated byincreased endothelial immunostaining and increased deposition ofradiolabelled antibody in the ischemic territory. In the lattertechnique, antibody deposition into the ischemic hemisphere wasnormalized to that in the nonischemic hemisphere in each animal, notonly to minimize potential variations in injection volume or volume ofdistribution, but to enable comparison between animals given differentantibodies. Because disruption of the endothelial barrier function inthe ischemic cortex may augment nonselective antibody deposition,similar experiments were performed with a control rat IgG. These datashow that the antibody which binds to P-selectin is deposited at anaccelerated rate compared with the control antibody, suggesting thatlocal P-selectin expression is augmented in the reperfused tissue. Thisdata in the murine model parallels that reported in a baboon model ofstroke¹⁰, in which P-selectin expression was increased within 1 hourfollowing the ischemic event.

The role of P-selectin expression in recruiting PMNs to thepost-ischemic zone was demonstrated using a strategy in whichaccumulation of ¹¹¹In-labelled PMNs was measured. Although we havepreviously reported that by 22 hours, PMN accumulation is elevated inthe ischemic hemisphere⁷, the current time-course data demonstrate thatPMN accumulation begins shortly after the onset of ischemia. Failure toexpress the P-selectin gene was associated with reduced PMNaccumulation, suggesting the participation of P-selectin inpost-ischemic cerebral PMN recruitment. However, the P-selectin nullanimals did demonstrate a modest (albeit less than control) neutrophilaccumulation by 22 hours. This data indicates that P-selectin is not theexclusive effector mechanism responsible for postischemic cerebral PMNrecruitment, and is consistent with our previous data that ICAM-1 alsoparticipates in post-ischemic PMN adhesion⁷. Furthermore, this data isnot unlike that in which intra-abdominal instillation of thioglycollatein P-selectin deficient mice caused delayed (but not absent) PMNrecruitment⁹.

Because of the critical need to identify reasons for failed reperfusion,the current studies examined the role of P-selectin in delayedpostischemic cerebral hypoperfusion^(21,22), the phenomenon whereinblood flow declines during reperfusion, despite restoration of adequateperfusion pressures. In cardiac models of ischemia, no-reflow worsens astime elapses after reperfusion³⁵, suggesting an important role forrecruited effector mechanisms, such as progressive microrcirculatorythrombosis, vasomotor dysfunction, and PMN recruitment. Both P-selectinand ICAM-1-dependent adherence reactions³⁶ and PMN capillary plugging³⁷have been shown in other models to participate in post-ischemicno-reflow. In the brain, PMNs have been implicated in post-ischemiccerebral no reflow^(38,39), but the role of P-selectin had not beenpreviously elucidated.

The current study uses a relatively noninvasive technique (laserdoppler) to obtain serial measurements of relative cerebral blood flow,in order to establish the existence, time course, andP-selectin-dependence of post-ischemic cerebrovascular no-reflow. Inorder to demonstrate that the threading procedure itself was not thecause of vascular damage and subsequent cerebral infarction, shamischemia experiments were performed (n=10) in which a nylon suture wasthreaded into the internal carotid artery for a 45 minute non-occludingperiod. In these experiments, the threading was shown to be nonocclusivebased upon no decline in perfusion by laser doppler during the 45 minuteperiod. When brains were than collected and stained with TTC at 24hours, none showed evidence of cerebral infarction. Therefore, we canconclude that the threading procedure per se does not provoke sufficientdamage to affect our major outcome variables. When relative cerebralblood flow was examined following frank middle cerebral artery occlusionin experimental animals, we observed that P-selectin null and controlanimals were subjected to virtually identical degrees of ischemia (therewas an initial ≈4.5-fold drop in relative cerebral blood flow followingmiddle cerebral artery occlusion in both). However, there was a slightincrease in relative cerebral blood flow in the first 10 minutesfollowing occlusion, even though the occluding suture remained in place.This is an empiric observation we have consistently made, for whichthere are likely to be several possible explanations. There is likely tobe some degree of collateral flow which opens up in the ischemicterritory. Another tenable explanation is that there may be an elementof initial vasospasm in the region of the occluding catheter tip, whichmodestly resolves within several minutes. Although both of theseexplanations are possible, due to the small size of the murinevasculature, we cannot identify the mechanism with certainty in ourmodel. Nevertheless, as we observe the same degree of flow recruitmentin both control and experimental animals, these data do not alter ourmain conclusions, that P-selectin is an important mediator of cerebraltissue injury in reperfused stroke.

Following removal of the intraluminal occluding suture, instantaneousrecovery of blood flow was the same in both the P-selectin +/+ and −/−animals. The fact that flow levels never returned to baseline (nor wasthere an overshoot, as might be seen with reactive hyperemia) may be dueto the severity and duration of the ischemic period, which is likely torecruit other mechanisms of post-ischemic cerebrovascular no-reflow,such as thrombosis or neutrophil recruitment caused bynon-selectin-dependent mechanisms. When even later time points areexamined (such as 30 minutes to 22 hours after removal of the occludingsuture), it is interesting to note that there is a slight decline incerebral blood flow in the P-selectin −/− animals. This late (albeitlimited) decline in cerebral blood flow by 22 hours is consistent withthe modest PMN recruitment observed in the PS −/− animals over the sameperiod, again suggesting the recruitment of other flow-limiting effectormechanisms (such as ICAM-1) in the PS −/− animals.

The functional effects of P-selectin expression are clear from thecurrent set of studies: animals which fail to express the P-selectingene (or PS +/+ animals treated with a functionally blockinganti-P-selectin antibody) exhibit smaller infarcts and improved survivalcompared with controls. When these data are considered along withpreviously published data demonstrating a deleterious role for ICAM-1expression in stroke⁷, it becomes increasingly apparent that there aremultiple means for recruiting PMNs to post-ischemic cerebral cortex, andthat blockade of each represents a potential strategy to improve strokeoutcome in humans. Given our current recognition of the importance oftimely reperfusion in halting the advancing wavefront of neuronal deathfollowing stroke, interfering with PMN adhesion at its earliest stagesappears to be an attractive option for reducing morbidity and mortality.In fact, anti-adhesion molecule strategies may not only be beneficial intheir own right (i.e., including patients ineligible for thrombolysis),but may extend the window of opportunity for thrombolyticintervention⁴⁰. The current set of studies contributes to ourunderstanding of pathophysiological mechanisms operative in reperfusedstroke. These studies suggest the need for clinical trials of therapiesfor evolving stroke which optimize the reperfusion milieu to reduce PMNaccumulation.

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13. Dirnagl U, Kaplan B, Jacewicz M, Bulsinelli W: Continuousmeasurement of cerebral blood flow by laser-doppler flowmetry ion a ratstroke model. J Cereb Blood Flow Metab 1989;9:589-596

14. Ley K, Bullard D C, Arbones M L, Bosse R, Vestweber D, Tedder T F,Beaudet A L: Sequential contribution of L- and P-selectin to leukocyterolling in vivo. J Exp Med 1995;181:669-675

15. David G S, Reisfeld R A: Protein iodination with solid statelactoperoxidase. Biochem 1974;13:1014-1021

16. Bederson J B, Pitts L H, Nishimura M C, Davis R L, Bartkowski H M:Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain fordetection and quantification of experimental cerebral infarction inrats. Stroke 1986;17:1304-1308

17. Huang Z, Huang P L, Panahian N, Dalkara T, Fishman M C, Moskowitz MA: Effects of cerebral ischemia in mice deficient in neuronal nitricoxide synthase. Science 1994;265:1883-1885

18. Bosse R, Vestweber D: Only simultaneous blocking of the L- andP-selectin completely inhibits neutrophil migration into mouseperitoneum. Eur J Immunol 1994;24:3019-3024

19. Kunkel E J, Jung U, Bullard D C, Norman K E, Wolitzky B A, VestweberD, Beaudet A L, Ley K: Absence of trauma-induced leukocyte rolling inmice deficient in both P-selectin and ICAM-1. J Exp Med 1996;183:57-65

20. Springer T A: Adhesion receptors of the immune system. Nature1990;346:425-434

21. Ames A I, Wright R L, Kowada M, Thurston J M, Majno G: Cerebralischemia II: the no reflow-phenomenon. Am J Pathol 1968;52:437-447

22. Levy D E, Van Uitert R L, Pike C L: Delayed postischemichypoperfusion: a potentially damaging consequence of stroke. Neurology1979;29:1245-1252

23. Weiss S J: Tissue destruction by neutrophils. N Engl J Med1989;320(6):365-376

24. Hallenbeck J M, Dutka A J, Tanishima T, Kochanek P M, Kumaroo K K,Thompson C B, Obrenovitch T P, Contreras T J: Polymorphonuclearleukocyte accumulation in brain regions with low blood flow during theearly postischemic period. Stroke 1986;17:246-253

25. Kochanek P M, Hallenbeck J M: Polymorphonuclear leukocytes andmonocytes/macrophages in the pathogenesis of cerebral ischemia andstroke. Stroke 1992;23(9):1367-1379

26. Dutka A J, Kochanek P M, Hallenbeck J M: Influence ofgranulocytopenia on canine cerebral ischemia induced by air embolism.Stroke 1989;20:390-395

27. Bednar M M, Raymond S, McAuliffe T, Lodge P A, Gross C E: The roleof neutrophils and platelets in a rabbit model of thromboembolic stroke.Stroke 1991;22(1):44-50

28. Geng J-G, Bevilacqua M P, Moore K L, McIntyre T M, Prescott S M, KimJ M, Bliss G A, Zimmerman G A, McEver R P: Rapid neutrophil adhesion toactivated endothelium medicated by GMP-140. Nature 1990;343:757-760

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30. Winn R K, Liggitt D, Vedder N B, Paulson J C, Harlan J M:Anti-P-selectin monoclonal antibody attenuates reperfusion injury in therabbit ear. J Clin Invest 1993;92:2042-2047

31. Seekamp A, Till G O, Mulligan M S, Paulson J C, Anderson D C,Miyasaka M, Ward P A: Role of selectins in local and remote tissueinjury following ischemia and reperfusion. Am J Pathol 1994;144:592-598

32. Kubes P, Jutila M, Payne D: Therapeutic potential of inhibitingleukocyte rolling in ischemia/reperfusion. J Clin Invest1995;95:2510-2519

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EXAMPLE 11 Use of Carbon Monoxide To Treat An Ischemic Disorder—Exampleof the Protective Effects of Carbon Monoxide in Lung Ischemia

In the initial patent application, we revealed data indicating thatendogenous production of carbon monoxide or administration of exogenouscarbon monoxide is beneficial in protecting the brain against subsequentischemic injury. As another example of the use of carbon monoxide intreating an ischemic disorder, we have administered carbon monoxide torats to test its effects on improving lung preservation fortransplantation (this is similar to an ischemic disorder, because thedonor lungs are removed from a recipient; during the period in which thelungs are preserved and transferred from donor to recipient, there is aninterruption in blood flow).

Methods for testing the effect of Carbon Monoxide on Lung Preservation:

Materials used to prepare preservation solution:

For all experiments, the base preservation solution consisted ofmodified Euro-Collins (EC) solution (Na⁺ 10mEq/L, K⁺115 mEq/L, Cl⁻ 15mEq/L, HPO₄ ²⁻ 85 mEq/L, H2PO₄ ⁻ 15 mEq/L, HCO₃ ⁻ 10 mEq/L).

Lung harvest, preservation and transplantation:

Inbred male Lewis rats (250-300 gms) were used for all experimentsaccording to a protocol approved by the Institutional Animal Care andUse Committee at Columbia University, in accordance with guidelines setforth by the American Academy for Accreditation of Laboratory AnimalCare (AAALAC). Lung transplant experiments were performed in thefollowing manner. Donor rats were given 500 units of heparinintravenously, and the pulmonary artery (PA) was flushed with a 30 mLvolume of 4° C. preservation solution at a constant pressure of 20 mmHg. When lungs are preserved in this manner, most of the infused flushsolution comes out of a left atrial vent created in the lung donor, aswell as out of the pulmonary veins following transection.

The left lung was then harvested, a cuff was placed on each vascularstump, a cylinder was inserted into the bronchus, and the lung wassubmerged for 6 hours in 4° C. preservation solution which was identicalto the PA flush solution. Gender/strain/size matched rats wereanesthetized, intubated, and ventilated with 100% O₂ using a rodentventilator (Harvard Apparatus, South Natick, Mass.). Orthotopic leftlung transplantation was performed through a left thoracotomy using arapid cuff technique for all anastomoses, with warm ischemic timesmaintained below 5 minutes. The hilar cross-clamp was released,re-establishing blood flow and ventilation to the transplanted lung. Asnare was then passed around the right PA, and Millar catheters (2F;Millar Instruments, Houston, Tex.) were introduced into the main PA andthe left atrium (LA). A Doppler flow probe (Transonics, Ithaca, N.Y.)was placed around the main PA.

Measurement of lung graft function:

Online hemodynamic monitoring was accomplished using MacLab and aMacintosh IIci computer. Measured hemodynamic parameters included LA andPA pressures (mm Hg), and PA flow (mL/min). Arterial oxygen tension(pO₂, mm Hg) was measured during inspiration of 100% O₂ using a modelABL-2 gas analyzer (Radiometer, Copenhagen, Denmark). PVRs werecalculated as (mean PA pressure−LA pressure)/mean PA flow and expressedas mm Hg/mL/min. After baseline measurements, the native right PA wasligated and serial measurements taken every five minutes until the timeof euthanasia at 30 minutes (or until recipient death).

Administration of Carbon Monoxide:

At the indicated time before surgery (4,8, or 12 hours), rats wereplaced in a bell jar, and carbon monoxide was administered at variousconcentrations (0.01%, 0.03%, or 0.1%), with the remainder of the gasmixture consisting of room air. (The gas was passed through a jar ofwater prior to administration, in order to humidify it for animalcomfort). At the indicated times following initiation of exposure, ratswere anesthetized and lungs harvested as described above. These donorlungs were used in subsequent lung transplant experiments.

Results and Discussion:

The results of these experiments indicate that, compared with untreatedcontrols, the inhalation of carbon monoxide prior to lung harvestconfers significant protection for the lungs following transplantation.This protection is evidenced by; (1) Improved arterial oxygenation ofthe recipients of carbon-monoxide-pretreated donor lungs; (2) Increasedpulmonary arterial blood flow (and reduced pulmonary vascularresistance) with the use of carbon-monoxide-pretreated donor lungs; and(3) Improved survival of recipients of carbon-monoxide-pretreated donorlungs compared with controls. The beneficial effects of carbon monoxidewere dose-dependent, i.e., the best protection was seen at the 0.1%dose, with an intermediate level of protection seen at the 0.03% dose,and the least protection seen at the 0.01% dose. The beneficial effectsof carbon monoxide were also time-dependent, in that longer exposuresappeared to provide the greatest protection. Together, these dataindicate that carbon monoxide can protect in another ischemic disorder(lung ischemia) and suggest that the results may be generalizable toother ischemic disorders as well.

EXAMPLE 12 Use of a Spectrophotometric Hemoglobin Assay To ObjectivelyQuantify Intracerebral Hemorrhage in Mice

Abstract

Background and Purpose There is a great interest in developing novelanticoagulant or thrombolytic strategies to treat ischemic stroke.However, at present, there are limited means to accurately assess thehemorrhagic potential of these agents. The current studies were designedto develop and validate a method for accurately quantifying the degreeof intracerebral hemorrhage in murine models. Methods In a murine model,intracerebral hemorrhage (ICH) was induced by stereotacticintraparenchymal infusion of collagenase B alone (6×10⁻⁶ units, n=5) orcollagenase B followed by intravenous tissue plasminogen activator(rt-PA, 0.1 mg/kg, n=6). Controls consisted of either sham surgery withstereotactic infusion of saline (n=5) or untreated animals (n=5). ICHwas (1) graded by a scale based on maximal hemorrhage diameter oncoronal sections, and (2) quantified by a spectrophotometric assaymeasuring cyanomethemoglobin in chemically reduced extracts ofhomogenized murine brain. This spectrophotometric assay was validatedusing known quantities of hemoglobin or autologous blood added to aseparate cohort of homogenized brains. Using this assay, the degree ofhemorrhage following focal middle cerebral artery occlusion/reperfusionwas quantified in mice treated with post-occlusion high-dose IV rt-PA(10 mg/kg, n=11) and control mice subjected to stroke but treated withphysiological saline solution (n=9). Results Known quantities ofhemoglobin or autologous blood added to fresh whole brain tissuehomogenates showed a linear relationship between the amount added and ODat the absorbance peak of cyanomethemoglobin (r=1.00 and 0.98,respectively). When in vivo studies were performed to quantifyexperimentally-induced ICH, animals receiving intracerebral infusion ofcollagenase B had significantly higher ODs than saline-infused controls(2.1-fold increase, p=0.05). In a middle cerebral artery occlusion andrepersfusion model of stroke, administration of rt-PA after reperfusionincreased the OD by 1.8-fold compared with animals which receivedphysiological saline solution (p<0.001). When the two methods ofmeasuring ICH (visual scorer and OD) were compared, there was a linearcorrelation (r=0.88). Additional experiments demonstrated thattriphenyltetrazolium staining, which is commonly used to stain viablebrain tissue, does not interfere with the spectrophotometricquantification of ICH. Conclusions These data demonstrate that thespectrophotometric assay accurately and reliably quantifies murine ICH.This new method should aid objective assessment of the hemorrhagic risksof novel anticoagulant or thrombolytic strategies to treat stroke andcan facilitate quantification of other forms of intracerebralhemorrhage.

Introduction

Ischemic stroke accounts for the greatest majority of presentations inacute stroke. There has thus been a tremendous interest in designingstrategies which can promptly and effectively restore blood flow to theischemic region of brain. Although heparin may be effective in incipientstroke (TIAs)³, its use during the acute phases of stroke may beassociated with a high degree of morbidity and intracerebralhemorrhage¹⁻⁴. Similarly, in the early 1960s, the dismal outcomes in thestreptokinase trials for acute stroke led to the reluctance ofclinicians to thrombolyse acute stroke for the subsequent threedecades^(5,6). This reluctance has been validated by recent trials inwhich the use of streptokinase has been associated with increased riskof mortality and intracerebral hemorrhage⁷. On the other hand, the useof recombinant tissue-type plasminogen activator (rt-PA) to treatstroke-in-progress has shown more promise⁸, with a subset of patientswith acute stroke who are treated with rt-PA demonstrating reducedlong-term morbidity if treated within the first 3 hours of symptomonset⁹⁻¹¹. Even so, other trials using the same agent (rt-PA) havefailed to show benefit or have had excessively high rates ofICH^(9,12-15).

This confusing morass of clinical data underscores the urgent need toidentify improved strategies to achieve rapid reperfusion. Towards thisend, it is imperative to identify an experimental model in which thepotential benefits of timely reperfusion in stroke can be weighedobjectively against the risks of increased intracerebral hemorrhage. Inmost animal studies of thrombolytic therapy for clinical stroke, therisks of intracerebral hemorrhage have been estimated rather thanquantitatively measured¹⁶⁻²⁴. The current studies were designed todevelop and validate a method for accurately quantifying the degree ofintracerebral hemorrhage in murine models, in order to assess potentialrisks of new anticoagulant or thrombolytic treatments for acute stroke.

Materials and Methods

Experimental Animals

In the present study, male C57BL/6J mice were purchased from JacksonLaboratories (Bar Harbor, Me.), and were used between 8 to 10 weeks old(22-32 g). All procedures were performed according to an institutionallyapproved protocol and are in accordance with the guidelines provided bythe American Academy of Accreditation of Laboratory Animal Care(AAALAC).

Spectrophotometric assay for intracerebral hemorrhage

The hemoglobin content of brains subjected to the experimentalprocedures below was quantified using a spectrophotometric assay asfollows. Whole brain tissue was obtained from freshly euthanized controlor experimental animals, and each brain was treated individually asfollows. Distilled water (250 μl) was added to each brain, followed byhomogenization for 30 sec (Brinkman Instruments, Inc., Westbury, N.Y.),sonication on ice with a pulse ultrasonicator for 1 minute (SmithKlineCorporation, Collegeville, Pa.), and centrifugation at 13,000 rpm for 30minutes (Baxter Scientific Products, Deerfield, Ill.). After collectingthe hemoglobin-containing supernatant, 80 μl of Drabkin's reagent(purchased from Sigma Diagnostics, St. Louis, Mo.; K₃Fe(CN)₆ 200 mg/L,KCN 50 mg/L, NaHCO₃ 1 g/L, pH 8.6²⁵) was added to a 20 μl aliquot andallowed to stand for 15 minutes. This reaction converts hemoglobin tocyanomethemoglobin, which has an absorbance peak at 540 nm, and whoseconcentration can then be assessed by the optical density of thesolution at ≈550 nm wavelength²⁶. To validate that the measuredabsorbance following these procedures reflects the amount of hemoglobin,known quantities of bovine erythrocyte hemoglobin (Sigma, St. Louis,Mo.) were analyzed using similar procedures alongside every brain tissueassay. As an additional measure blood was obtained from control mice bycardiac puncture following anesthesia. Incremental aliquots of thisblood were then added to freshly homogenized brain tissue obtained fromuntreated mice to generate a standard absorbance curve.

Collagenase-induced intracerebral hemorrhage

The general procedures for inducing intracerebral hemorrhage in themouse were adapted from a method which has been previously described inrats²⁷. After anesthesia with an intraperitoneal injection of 0.35 ml ofketamine (10 mg/ml) and xylazine (0.5 mg/ml), mice were positioned pronein a stereotactic head frame. The calvarium was exposed by a midlinescalp incision from the nasion to the superior nuchal line and then theskin was retracted laterally. Using a variable speed drill (Dremel,Racine, Wis.) a 1.0-mm burrhole was made 2.0 mm posterior to the bregmaand 2.0 mm to the right of midline. A single 22-gauge angiocatheterneedle was inserted using stereotactic guidance into the right deepcortex/basal ganglia (coordinates: 2.0 mm posterior, 2.0 mm lateral).The needle was attached by a plastic tubing to a microinfusion syringeand solutions were infused into the brain at a rate of 0.25 μl perminute for 4 minutes with an infusion pump (Bioanalytical Systems, WestLafayette, Ind.). Animals received either: (1) 0.024 μg collagenase B(Boehringer Mannheim, Mannheim, Germany) in 1 μl normal saline solution(Collagenase); (2) 1 μl normal saline solution alone (Sham); (3) notreatment (Control); or (4) stereotactically-guided infusion ofcollagenase B as above but followed immediately by intravenousrecombinant human tissue plasminogen activator (Genentech Inc., SouthSan Francisco, Calif., 1 mg/kg in 0.2 ml normal saline solution)administered by dorsal penile vein injection (Collagenase+rt-PA). In theCollagenase, Sham, and Collagenase+rt-PA groups, the stereotactic needlewas removed immediately following fusion and the incision was closedwith surgical staples. Brain tissue was harvested immediately afterrapid anesthetized decapitation.

Hemorrhagic conversion in a murine focal cerebral ischemia model

Focal cerebral ischemia was produced in animals by transient rightmiddle cerebral artery occlusion using a method previously described indetai^(28,29). Briefly, a heat-blunted 12 or 13 mm 5-0 or 6-0 gaugenylon suture was passed into the right internal carotid artery to thelevel of the middle cerebral artery. After 45 minutes, the occludingsuture was removed to reestablish perfusion. Immediately followingremoval of the occluding suture, animals received either intravenoustissue plasminogen activator (10 mg/kg in 0.2 ml normal saline solution,Stroke+rt-PA) or normal saline solution (Stroke+Saline) given by dorsalpenile vein injection. At 24 hours, brain tissue was harvestedimmediately after rapid anesthetized decapitation. To evaluate theeffect of 2,3,5-triphenyltetrazolium chloride (TTC), which is commonlyused to distinguish infarcted from noninfarcted cerebral tissue^(28,30),nonmanipulated (control) brains were divided into half, immersed inimmersed in 2% TTC (Sigma Chemical Company, St. Louis, Mo.) in 0.9%phosphate-buffered saline, incubated for 30 minutes at 37° C., and thenprepared as described above for the spectrophotometric hemoglobin assay.The other half of each brain was immersed in saline for an identicalduration, and then subjected to the procedures described above for thespectrophotometric hemoglobin assay.

Validation of quantitative intracerebral hemorrhage assay

The degree of ICH was first scored visually by a blinded observer. Forvisual scoring of intracerebral hemorrhage in mice, brains obtained frommice which had survived to the 24 hour time point following theprocedure (collagenase-induced hemorrhage or MCA occlusion) were placedin a mouse brain matrix (Activational Systems Inc., Warren, Mich.) toobtain 1 mm serial coronal sections. Sections were inspected by ablinded observer and brains were given an ICH score from a graded scalebased on maximal hemorrhage diameter seen on any of the sections [ICHscore 0, no hemorrhage; 1, <1 mm; 2, 1-2 mm; 3, >2-3 mm; 4, >3 mm].Slices from each brain were then pooled, homogenized, and then treatedaccording to the procedures described above for the spectrophotometrichemoglobin assay.

Statistics

Correlations between visually-determined ICH scores andspectrophotometric determinations of ICH were performed using Pearson'slinear correlation, with correlation coefficients indicated. Toestablish whether a given treatment (Collagenase, Sham, Stroke, etc.)had a significant effect on either spectrophotometric or visually-scoredICH, comparisons were made using an unpaired two-tailed t-test. Fornonparametric data (visual ICH scores), nonparametric analysis wasperformed using the Mann-Whitney test. Values are expressed as means±SEM, with a p<0.05 considered statistically significant.

Results

Spectrophotometric hemoglobin assay

Initial studies were performed to determine the reliability andreproducibility of the spectrophotometric hemoglobin assay. In the firstset of experiments, known quantities of hemoglobin were converted tocyanomethemoglobin according to previously published procedures, and theOD measured [FIG. 35A]²⁶. In a second set of experiments, knownquantities of autologous blood were added to fixed volumes of freshbrain tissue homogenate, with further treatment of specimens asdescribed above. These data show that the optical density ofcyanomethemoglobin-containing supernatants at 550 nm correlated linearlywith the amount of added blood [FIG. 35B]. These data show tight linearcorrelation (r=1.00 and 0.98 for FIGS. 35A and 35B, respectively), aswell as excellent reproducibility as gauged by relatively small standarderrors of the mean. To establish the TTC (commonly used to distinguishinfarcted from noninfarcted cerebral tissue^(28,30)) does not affect thespectrophotometric hemoglobin assay, nonmanipulated (control) brainswere divided into half, with half being subjected to the standard TTCstaining procedure and half being treated with saline as a control.These data (compare FIG. 35B, solid and dashed lines) indicate thatpretreatment of brain tissue with TTC does not affect thespectrophotometric hemoglobin assay.

To determine whether this method is able to detect ICH, the assay wasperformed on murine intracerebral hemorrhage caused by two differentprocedures, intraparenchymal collagenase infusion or middle cerebralartery occlusion/reperfusion. In the first procedure, collagenase B wasapplied as a local infusion through a burrhole, in order to weaken thevascular wall to promote ICH (Collagenase group). To further increasethe propensity for and degree of ICH, a similar procedure was performed,with immediate administration of rt-PA following the procedure(Collagenase+rt-PA group). Two control conditions were also included, asham operation which included drilling the burrhole but withinstillation of physiological saline (Sham), and an untreated group(Control). These experiments demonstrated that collagenase infusionincreases the amount of intracerebral blood detected by thespectrophotometric assay (especially with collagenase+rt-PA) comparedwith sham-treated animals or normal controls [FIG. 36A].

In the second and perhaps more clinically relevant method for inducingICH, a stroke was created by transient intraluminal occlusion of themiddle cerebral artery followed by reperfusion. In addition, weattempted to increase the propensity for hemorrhagic conversion byadministration of a thrombolytic agent. Two groups were studied, thosewhich had received normal saline solution or those which receivedintravenous rt-PA immediately following removal of the intraluminaloccluding suture. These data indicate that the addition of afibrinolytic agent following stroke increases the amount of ICH which isdetected by the spectrophotometric hemoglobin assay [FIG. 36B]. It isinteresting to note that baseline absorbance is lower in animalssubjected to stroke than control/untreated animals [FIGS. 36A and 36B].To further investigate how residual intravascular blood might affect thespectrophotometric hemoglobin assay, experiments were performed inwhich, immediately prior to decapitation of the animal for brainharvest, a cephalic perfusion of physiological saline was performed(administered via the left cardiac ventricle). In control animals (n=5),which recieved cardiac saline perfusion prior to brain harvest, the meanOptical Density following tissue preparation and spectrophotometrichemoglobin assay was 0.25±0.3 (this is lower than the Optical densityseen in non-cardiac perfused animals subjected to either no or shamsurgery (n=10, OD 0.34±0.05, p=0.05 vs cardiac perfused controls). Onthe other hand, following stroke, there was no difference in O.D.whether or not cardiac saline perfusion was performed (0.15±0.04 forstroke without cardiac saline perfusion, n=5; 0.15±0.03 for stroke withcardiac saline perfusion, P=NS). When saline-perfused animals withstroke were compared to saline-perfused animals without stroke, there isan apparent reduction in OD following spectrophotometric hemoglobinassay. These data would suggest that animals with a stroke have lessintracerebral blood detected, perhaps as the result of a reduction ofthe total amount of blood in the ipsilateral MCA following ischemia.

Visual ICH score

In order to further validate the spectrophotometric hemoglobin assay, wecompared it to morphometric assessment of hemorrhage size, which hastraditionally been used in the literature³¹⁻³⁵. We developed a visualscoring system (0-4) in which a blinded observer scored the degree ofICH in serial cerebral sections based upon maximal hemorrhage diameter.This visual assessment was performed on a photograph of the brain takenimmediately prior to the performance of the spectrophotometrichemoglobin assay [FIG. 37], so that the two techniques could becorrelated on the same specimens. When compared with controls notsubjected to any intervention, animals receiving a sham local infusion(i.e., burrhole+saline) demonstrate only a slight increase in visual ICHscore [FIG. 38A]. However, when either collagenase alone orcollagenase+rt-PA was added to the infusate, visual ICH scores weresignificantly increased [FIG. 38A]. In the stroke model, rt-PA similarlyresulted in an increase in the visual ICH score [FIG. 38B]. When thedata are plotted to show the relationship between the visual ICH scoreand the spectrophotometric technique for quantifying ICH, a linearrelationship was suggested (r=0.88), however, with smaller degrees ofhemorrhage (visual ICH scores of 0 or 1), this relationship did not hold[FIG. 39].

Discussion

Recently, it has become apparent that early intervention in stroke withcertain intravenous thrombolytic agents (rt-PA) may be beneficial ifinstituted within 3 hours of symptom onset^(9,10). However,administration of thrombolytic agents outside of this narrow therapeuticwindow can cause an unacceptably high incidence of devastating ICH(streptokinase vs. placebo, 10 day mortality 34.0% vs. 18.2%, p=0.002, 6month mortality 73% vs. 59%, p=0.06)⁷. It therefore remains a clinicalimperative to identify more optimal agents for restoring perfusion whichare associated with less risk of hemorrhagic conversion. In order toadequately study new agents which interfere with coagulation orfibrinolytic mechanisms, it is necessary to have an objective means ofquantifying the downside risk of ICH. In the experimental literature,quantification of ICH has been performed either by radiological imagingprocedures³²⁻³⁷, or by a visual estimation of the amount of hemorrhagein postmortem brain tissue ³¹⁻³⁵. These procedures are of limitedusefulness depending on the conditions under study. For instance, inaddition to the logistic constraints imposed by the need forsophisticated equipment, most radiological imaging techniques are oflimited use in murine models, which may preclude their use in theevaluation of transgenic mice, a potentially powerful tool for studyingthe coagulation or fibrinolytic systems. Visual estimation of ICH issubjective in nature, and as our own data show, may be relativelyinsensitive for detecting small degrees of ICH. Furthermore, neither theradiological nor the visual techniques permit accurate quantification ofICH when the hemorrhagic region is patchy or multifocal.

The current studies were performed to develop and validate an objectivemethod for quantifying ICH in experimental animals. The use of aspectrophotometric assay for the quantification of hemoglobin based uponthe conversion of hemoglobin to cyanomethemoglobin has been previouslyreported^(26,31). However, to the best of our knowledge, in the brain,it has only been used in rats to measure the size of a frank blood clotfollowing its removal from adjacent brain tissue³¹. Thespectrophotometric assay we describe and validate can be used in animalsas small as mice, which facilitates the use of the many transgenic mousestrains now available (particularly those with alterations in thethrombotic or fibrinolytic cascades). Furthermore, thisspectrophotometric assay permits the quantification of ICH even whenthere are patchy or multifocal hemorrhages, which could be otherwisedifficult to identify or isolate. Finally, in contrast to the Lee study,we have validated our study for reproducibility and reliability usingknown quantities of hemoglobin and autologous blood admixed with braintissue³¹. Because the surgical procedure used in the stroke experimentsdid not significantly alter blood hemoglobin concentrations (data notshown), the spectrophotometric hemoglobin assay may be used toextrapolate the volume of intracerebral hemorrhage when the hemoglobinconcentration is known at the time of hemorrhage.

To develop and validate the spectrophotometric hemoglobin assay forsituations that may be relevant for clinical ICH, we createdintracerebral hemorrhages by two different methods: (1) intracerebralinjection of collagenase (to weaken the vascular wall, as might occurwith an aneurysm or with trauma; and (2) in a model of stroke. In bothinstances, a cohort of animals also received rt-PA, in order to validatethe model at the high end of the spectrum of ICH. Because there has beenno established gold-standard measurement for ICH in mice, ourspectrophotometric measurements were compared to ICH size asindependently assessed by visual scoring. Finally, to prove the assayeven more useful for experimental models of stroke in which brains arestained with triphenyltetrazolium chloride (TTC) to quantify cerebralinfarct volume, the brains of animals subjected to MCAocclusion/reperfusion were stained with TTC prior to pooling andhomogenization to establish that the TTC staining procedure itself doesnot interfere with the ability to quantify ICH by the spectrophotometrichemoglobin assay. These data [FIG. 1B] indicate that there is nodetectable cross-interference between the two procedures when usedsequentially (TTC staining first, followed by homogenization and thespectrophotometric hemoglobin assay).

In addition to its stability to detect ICH, the current studies indicatethat this technique may also given an indication of the amount ofresidual intravascular blood following brain harvest. The procedure ofcephalic saline perfusion does not alter the optical density forcyanomethemoglobin in brain subjected to stroke, suggesting that theamount of intravascular blood is relatively fixed and does not wash outby the procedure. However, in control animals who have been otherwiseuntreated, the saline perfusion treatment does appear to lower theoptical density for cyanomethemoglobin by about 30%. Our experiments donot provide the reason for this difference, but one may speculate thatfollowing stroke, there is an element of vasoconstriction/vaso-occlusionin the territory of infarction, which makes the saline perfusiontechnique less effective at washing out additional residualintravascular blood. Also, if there is truly an element ofvasoconstriction following stroke or experimentally-inducedintracerebral hemorrhage, this may reduce the intravascular blood pooland hence account for an overall lowering of the optical density whencontrol and stroke/ICH brains are compared (even if some extravascularblood is present in the latter group).

Several technical aspects of the spectrophotometric technique formeasuring intracerebral hemorrhage also deserve mention. For the currentexperiments, although there is a broad absorbance peak forcyanomethemoglobin centered around 540 nm, we measured the absorbance ofcyanomethemoglobin at 550 nm. The reason for doing this is that manyspectrophotometers have fixed wavelength capabilities depending upon thepreset filters, and 550 nm is a commonly used wavelength (especially inELISA plate readers). Although perhaps measurement of absorbance at 540nm would have yielded slightly higher optical density measurements, theabsorbance peak of cyanomethemoglobin is broad in this area, and hence550 nm may be used without the need to correct for the absorbance offerri- or ferrocyanide (the extinction coefficients forcyanomethemoglobin at 551 nm and 540 nm are 11.5 and 11.1, respectively,compared with the 41-fold lower extinction coefficient of ferri- orferrocyanide³⁸). Studies using a continuous wavelength spectrophotometer(which was used to measure OD at 540 nm) and a discrete spectrum ELISAplate reader (used to measure OD at 550 nm) gave similar results. As thelatter technique was simpler, increased the throughput of the procedure,and permitted us to minimize sample volume, we elected to use the lattertechnique for the studies shown in the Results section.

There are some potential other technical considerations that should beconsidered when using the spectrophotometric assay. Even though we haveshown that the spectrophotometric procedure can be used in conjunctionwith TTC staining of serial cerebral sections for infarct volumeanalysis, the tissue must be subsequently homogenized and extracted,destroying tissue architecture and making further histologicalcharacterization impossible. It is possible that this technique mayoverestimate the degree of ICH if extracerebral blood is unintentionallyincluded during brain harvesting, or the technique may underestimate thedegree of ICH if residual epidural, subdural, or subarachnoid bloodremains adherent to the calvarium, which is discarded during the processof brain removal.

Because of the nature of the measurement technique, in which light at agiven wavelength is absorbed along a fixed length path, anything causingturbidity of the homogenized brain supernatant may increase the ODreading. This may include lipids, abnormal plasma proteins, anderythrocyte stroma. In fact, in preliminary experiments, we found thatODs were falsely elevated when the centrifugation was insufficient andsome of the lipid layer was included in the assay. Free pyridines mayalter the absorbance spectrum of cyanomethemoglobin, and there is thepotential for other hemochromogens to also react with the Drabkin'sreagent³⁹. However, to our knowledge, these reactions should notinterfere to a significant extent with the determination ofintracerebral blood/hemorrhage.

In summary, the current data illustrate how a simple and inexpensivespectrophotometric assay for hemoglobin can provide a useful method forquantifying ICH. This technique should prove especially useful toevaluate the hemorrhagic potential of newly developed thrombolytic oranticoagulant therapies for the treatment of stroke.

References

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2. Babikian V L, Kase C S, Pessin M S, Norrving B, Gorelick P B:Intracerebral hemorrhage in stroke patients anticoagulated with heparin.Stroke 1989;20:1500-1503

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6. Meyer J S, Gilroy J, Barnhart M I, Johnson J F: Anticoagulants plusstreptokinase therapy in progressive stroke. JAMA 1964;189:373

7. Hommel M, Cornu C, Boutitie F, Boissel J P, The MultiCenter AcuteStroke Trial—Europe Study Group: Thrombolytic therapy with streptokinasein acute ischemic stroke. N Engl J Med 1996;335:145-150

8. Wardlaw J M, Warlow C P: Thrombolysis in acute ischemic stroke: doesit work? Stroke 1992;23:1826-1839

9. The National Institute of Neurological Disorders and Stroke rt-PAStroke Study Group: Tissue plasminogen activator for acute ischemicstroke. N Engl J Med 1995;333:1581-1587

10Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von Kummer R, BoysenG, Bluhmki E, Hoxter G, Mahagne M H, Hennerici M, for the ECASS StudyGroup: Intravenous thrombolysis with recombinant tissue plasminogenactivator for acute hemispheric stroke. J A M A 1995;274(13):1017-1025

11Trouillas P, Nighoghossian N, Getenet J C, Riche G, Neuschwander P,Froment J C, Turjman F, Jin J X, Malicier D, Fournier G, Gabry A L,Ledoux X, Derex L, Berthezene Y, Adeleine P, Xie J, Ffrench P,Dechavanne M: Open trial of intravenous tissue plasminogen activator inacute carotid territory stroke. Stroke 1996;27:882-890

12. Haley E C, Jr., Levy D E, Brott T G, Sheppard G L, Wong M C,Kongable G L, Torner J C, Marler J R: Urgent therapy for stroke, PartII. Pilot study of tissue plasminogen activator administered 91-180minutes from onset. Stroke 1992;23:641-645

13. Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von Kummer, R,Boysen G, Bluhmki E, Hoxter G, Mahagne M H, et al: Intravenousthrombolysis with recombinant tissue plasminogen activator for acutehemispheric stroke. The European Cooperative Acute Stroke Study (ECASS).JAMA 1995;274:1017-1025

14. Brott T G, Haley E C,Jr., Levy D E, Barsan W, Broderick J, SheppardG L, Spilker J, Kongable G L, Massey S, Reed R, et al: Urgent therapyfor stroke. Part I. Pilot study of tissue plasminogen activatoradministered within 90 minutes. Stroke 1992;23:632-640

15. del Zoppo G J, Poeck K, Pessin M S, Wolpert S M, Furlan A J, FerbertA, Alberts M J, Zivin J A, Wechsler L, Busse O, et al: Recombinanttissue plasminogen activator in acute thrombotic and embolic stroke.Annals of Neurology 1992;32:78-86

16. de Courten-Myers G M, Kleinholz M, Holm P, DeVoe G, Schmitt G,Wagner K R, Myers R E: Hemorrhagic infarct conversion in experimentalstroke. Ann Emerg Med 1992;21:120-126

17. Overgaard K, Sereghy T, Pedersen H, Boysen G: Neuroprotection withNBQX and thrombolysis with rt-PA in rat embolic stroke. NeurologicalResearch 1993;15:344-349

18. Overgaard K, Sereghy T, Boysen G, Pedersen, Diemer N H: Reduction ofinfarct volume by thrombolysis with rt-PA in an embolic rat strokemodel. Scandinavian Journal of Clinical & Laboratory Investigation1993;53:383-393

19. Benes, V, Zabramski J M, Boston M, Puca A, Spetzler R F: Effect ofintra-arterial tissue plasminogen activator and urokinase on autologousarterial emboli in the cerebral circulation of rabbits. Stroke1990;21:1594-1599

20. Overgaard K, Sereghy T, Pedersen H, Boysen G: Effect of delayedthrombolysis with rt-PA in a rat embolic stroke model. Journal ofCerebral Blood Flow & Metabolism 1994;14:472-477

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24. Lyden P D, Zivin J A, Soll M, Sitzer M, Rothrock J F, Alksne J:Intracerebral hemorrhage after experimental embolic infarction.Anticoagulation. Arch Neurol 1987;44:848-850

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28. Connolly E S Jr, Winfree C J, Springer T A, Naka Y, Liao H, Yan S D,Stern D M, Solomon R A, Gutierrez-Ramos J-C, Pinsky D J: Cerebralprotection in homozygous null ICAM-1 mice after middle cerebral arteryocclusion. Role of neutrophil adhesion in the pathogenesis of stroke. JClin Invest 1996;97:209-216

30. Bederson J B, Pitts L H, Nishimura M C, Davis R L, Bartkowski H M:Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain fordetection and quantification of experimental cerebral infarction inrats. Stroke 1986;17:1304-1308

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33. Qian L, Nagaoka T, Ohno K, Tominaga B, Nariai T, Hirakawa K, KuroiwaT, Takakuda K, Miyairi H: Magnetic resonance imaging and pathologicstudies on lateral fluid percussion injury as a model of focal braininjury in rats. Bulletin of Tokyo Medical & Dental University1996;43:53-66

34. Brown M S, Kornfeld M, Mun-Bryce S, Sibbitt R R, Rosenberg G A:Comparison of magnetic resonance imaging and histology incollagenase-induced hemorrhage in the rat. Journal of Neuroimaging1995;5:23-33

35. Thulborn K R, Sorensen A G, Kowall N W, McKee A, Lai A, McKinstry RC, Moore J, Rosen B R, Brady T J: The role of ferritin and hemosiderinin the MR appearance of cerebral hemorrhage: a histopathologicbiochemical study in rats. American Journal of Neuroradiology1990;11:291-297

36. Elger B, Seega J, Brendel R: Magnetic resonance imaging study on theeffect of levemopamil on the size of intracerebral hemorrhage in rats.Stroke 1994;25:1836-1841

37. Weingarten K, Zimmerman R D, Deo-Narine V, Markisz J, Cahill P T,Deck M D: MR imaging of acute intracranial hemorrhage: findings onsequential spin-echo and gradient-echo images in a dog model. AmericanJournal of Neuroradiology 1991;12:457-467

38. Drabkin D L, Austin J H: Spectrophotometric studies: II.Preparations from washed blood cells; nitric oxide hemoglobin andsulfhemoglobin. J Biol Chem 1935;112:51-65

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EXAMPLE 12 Active-site Blocked Factor IXa Limits MicrovascularThrombosis and Cerebral Injury In Murine Stroke Without IncreasingIntracerebral Hemorrhage

Summary

The clinical dilemma in stroke treatment is that agents which restorevascular patency increase the risk of intracerebral hemorrhage.Active-site blocked Factor IXa (IXai), formed from purified factor IXaby dansylation of its active site, competes with native Factor IXa toinhibit assembly of Factor IXa into the intrinsic Factor X activationcomplex. When pretreated with Factor IXai, mice subjected to focalcerebral ischemia and reperfusion demonstrated reduced microvascularfibrin and platelet deposition, increased cerebral reperfusion, andsignificantly smaller cerebral infarcts than vehicle-treated controls.Factor IXai-mediated cerebroprotection was dose-dependent, notassociated with intracerebral hemorrhage at therapeutically effectivedoses, and was seen even when Factor IXai was administered after theonset of cerebral ischemia.

Administration of Factor IXai represents a new strategy to treat strokein evolution without increasing the risk of intracerebral hemorrhage.

Introduction

Timely reestablishment of blood flow to ischemic brain represents thecurrent treatment paradigm for acute stroke¹⁻³. Administration of athrombolytic agent, even when given under optimal conditions, may notachieve this desired clinical result. Perfusion often fails to return topreischemic levels (postischemic hypoperfusion), suggesting thatischemic injury is not produced solely by the original occlusion, butthat there is also an element of microcirculatory failure. In addition,thrombolysis of acute stroke is associated with an increased risk ofintracerebral hemorrhage (ICH)¹⁻⁴, indicating that there remains a clearneed to identify new agents which can promote reperfusion withoutincreasing the risk of ICH.

Following an ischemic event, the vascular wall is modified from itsquiescent, anti-adhesive, antithrombotic state, to one which promotesleukocyte adhesion and thrombosis. In acute stroke, active recruitmentof leukocytes by adhesion receptors expressed in the ipsilateralmicrovasculature, such as ICAM-1⁵ and P-selectin⁶, potentiatespostischemic hypoperfusion. However, experiments with mice deletionallymutant for each of these genes demonstrate that even in their absence,postischemic cerebral blood flow (CBF) returns only partially tobaseline, suggesting the existence of additional mechanisms responsiblefor postischemic cerebrovascular no-reflow. To explore this possibility,the first set of experiments was designed to test the hypothesis thatlocal thrombosis occurs at the level of the microvasculature (distal tothe site of primary occlusion) in stroke.

To assess the deleterious consequences of microvascular thrombosis instroke, the second set of experiments tested the hypothesis thatselective blockade of the intrinsic pathway of coagulation could limitmicrovascular thrombosis, thereby protecting the brain in stroke. Thestrategy of selective inhibition of the intrinsic pathway of coagulationwas chosen because it is primarily responsible for intravascularthrombosis. Heparin, hirudin, and fibrinolytic agents interfere with thefinal common pathway of coagulation to inhibit the formation oraccelerate the lysis of fibrin, and therefore increase the propensityfor ICH. We hypothesized that selective blockade of IXa/VIIIa/Xactivation complex assembly might provide a novel mechanism to limitintravascular thrombosis while preserving mechanisms of extravascularhemostasis by the extrinsic/tissue factor pathway of coagulation whichmay be critical in infarcted brain tissue or adjacent regions wheresmall vessels are friable and subject to rupture. We used a novelstrategy in which a competitive inhibitor of Factor IXa (active-siteblocked IXa, or IXai) was given to mice subjected to stroke to test thehypothesis that is would improve stroke outcome without increasing ICH.

Methods

Murine stroke model: Transient focal cerebral ischemia was induced inmice by intralumenal occlusion of the middle cerebral artery (45minutes) and reperfusion (22 hrs) as previously reported⁷. Serialmeasurements of relative cerebral blood flow (CBF) were recorded vialaser doppler flowmetry⁷, and infarct volumes (% ipsilateral hemisphere)determined by planimetric/volumetric analysis of triphenyl tetrazoliumchloride (TTC)-stained serial cerebral sections⁷.

¹¹¹Indium-platelet studies: Platelet accumulation was determined using¹¹¹Indium labeled platelets, collected and prepared as previouslydescribed⁸. Immediately prior to surgery, mice were given 5×10⁶¹¹¹In-labeled-platelet intravenously; deposition was quantified after 24hours by as ipsilateral cpm/contralateral cpm.

Fibrin immunoblotting/immunostaining: The accumulation of fibrin wasmeasured following sacrifice (of fully heparinized animals) usingimmunoblotting/immunostaining procedures which have been recentlydescribed and validated⁹. Because fibrin is extremely insoluble, braintissue extracts were prepared by plasmin digestion, then applied to astandard SDS-polyacrylamide gel for electrophoresis, followed byimmunoblotting using a polyclonal rabbit anti-human antibody prepared togamma—gamma chain dimers present in cross-linked fibrin which can detectmurine fibrin, with relatively little cross-reactivity withfibrinogen¹⁰. Fibrin accumulation was reported as an ipsilateral tocontralateral ratio. In additional experiments, brains were embedded inparaffin, sectioned, and immunostained using the same anti-fibrinantibody.

Spectrophotometric hemoglobin assay and visual ICH score: ICH wasquantified by a spectrophotometric-based assay which we have developedand validated^(11,12). In brief, mouse brains were homogenized,sonicated, centrifuged, and methemoglobin in the supernatants converted(using Drabkin's reagent) to cyanomethemoglobin, the concentration ofwhich was assessed by measuring O.D. at 550 nm against a standard curvegenerated with known amounts of hemoglobin. Visual scoring of ICH wasperformed on 1 mm serial coronal sections by a blinded observer based onmaximal hemorrhage diameter seen on any of the sections [ICH score 0, nohemorrhage; 1, <1 mm; 2, 1-2 mm; 3, >2-3 mm; 4, >3 mm].

Preparation of Factor IXai¹³; Factor IXai was prepared by selectivelymodifying the active site histidine residue on Factor IXa, usingdansyl-glu-gly-arg-chloromethylkentone. Proplex was applied to apreparative column containing immobilized calcium-dependent monoclonalantibody to Factor IX. The column was washed, eluted withEDTA-containing buffer, and Factor IX in the eluate (confirmed as asingle band on SDS-PAGE) was then activated by applying Factor IXa(incubating in the presence of CaCl₂). Purified Factor IXa was reactedwith a 100-fold molar excess of dansyl-glu-gly-arg chloromethylketone,and the mixture dialyzed. The final product (IXai), devoid ofprocoagulant activity, migrates identically to IXa on SDS-PAGE. Thismaterial (Factor IXai) was then used for experiments followingfiltration (0.2 μm) and chromatography on DeToxi-gel columns, to removeany trace endotoxin contamination (in sample aliquots, there was nodetectable lipopolysaccharide). IXai was subsequently frozen intoaliquots at −80° C. until the time of use. For those experiments inwhich IXai was used, it was given as a single intravenous bolus at theindicated times and at the indicated doses.

Results

To crate a stroke in a murine model, a suture is introduced into thecerebral vasculature so that it occludes the orifice of the right middlecerebral artery, rendering the subtended territory ischemic. Bywithdrawing the suture after a 45 minute period of occlusion, areperfused model of stroke is created; mice so treated demonstrate focalneurological deficits as well as clear-cut areas of cerebral infarction.Because the occluding suture does not advance beyond the major vasculartributary (the middle cerebral artery), this model provides an excellentopportunity to investigate “downstream” events that occur within thecerebral microvasculature in response to the period of interrupted bloodflow. Using this model, the role of microvascular thrombosis wasinvestigated as follows. To demonstrate that platelet-rich thromboticfoci occur within the ischemic cerebral hemisphere, ¹¹¹In-labeledplatelets were administered to mice immediately prior to theintroduction of the intraluminal occluding suture, to track theirdeposition during the ensuing period of cerebral ischemia andreperfusion. In animals not subjected to the surgical procedure tocreate stroke, the presence of platelets was approximately equal betweenthe right and left hemispheres, as would be expected [FIG. 40A, leftbar]. However, when animals were subjected to stroke (and received onlyvehicle to control for subsequent experiments), radiolabeled plateletspreferentially accumulated in the ischemic (ipsilateral) hemisphere,compared with significantly less deposition in the contralateral(nonischemic) hemisphere [FIG. 40A, middle bar]. These data support theoccurrence of platelet-rich thrombi in the ischemic territory. WhenFactor IXai is administered to animals prior to introduction of theintraluminal occluding suture, there is a significant reduction in theaccumulation of radiolabelled platelets in the ipsilateral hemisphere[FIG. 40A, right bar].

Another line of evidence also supports the occurrence of microvascularthrombosis in stroke. This data comes from the immunodetection offibrin, using an antibody directed against a neoepitope on thegamma—gamma chain dimer of cross-linked fibrin. Immunoblots demonstratea band of increased intensity in the ipsilateral (right) hemisphere ofvehicle-treated animals subjected to focal cerebral ischemia andreperfusion [FIG. 40B, “Vehicle”]. In animals treated with Factor IXai(300 μg/kg) prior to stroke, there is no apparent increase in theipsilateral accumulation of fibrin [FIG. 40B, “Factor IXai”]. Todemonstrate that fibrin accumulation was due to the deposition ofintravascular fibrin (rather than due to nonspecific permeabilitychanges and exposure to subendothelial matrix), fibrin immunostainingclearly localized the increased fibrin to the lumina of ipsilateralintracerebral microvessels [FIG. 40C].

To investigate whether Factor IXai can limit intracerebral thrombosisand restore perfusion IXai was given to mice immediately prior to stroke(300 μg/kg). These experiments demonstrate both a reduction in¹¹¹In-platelet accumulation in the ipsilateral hemisphere [FIG. 41A] aswell as decreased evidence of intravascular fibrin by immunostaining.Furthermore, there is a significant increase in CBF by 24 hours,suggesting the restoration of microvascular patency by Factor IXai [FIG.41A]. The clinical relevance of this observation is underscored by theability of Factor IXai to reduce cerebral infarct volumes [FIG. 41B].These beneficial effects of Factor IXai were dose dependent, with 600μg/kg being the optimal dose [FIG. 41C].

Because the development of ICH is a major concern with any anticoagulantstrategy in the setting of stroke, the effect of IXai on ICH wasmeasured using our recently validated spectrophotometric method forquantifying ICH^(11,12). These data indicate that at the lowest doses(and the most effective ones), there is no significant increase in ICH[FIG. 42A]. At the highest dose tested (1200 μg/kg), there is anincrease in ICH, which was corroborated by a semiquantitative visualscoring method which we have also recently reported [FIG. 42B]^(11,12).

Because therapies directed at improving outcome from acute stroke mustbe given after clinical presentation, and because fibrin continues toform following the initial ischemic event in stroke, we tested whetherIXai might be effective when given following initiation of cerebralischemia. IXai given after middle cerebral artery occlusion (followingremoval of the occluding suture) provided significant cerebralprotection judged by its ability to significantly reduce cerebralinfarction volumes compared with vehicle-treated controls [FIG. 43].

Discussion

The data in these studies demonstrate clear evidence of intravascularthrombus formation (both platelets and fibrin) within the post-ischemiccerebral microvasculature. The pathophysiological relevance ofmicrovascular thrombosis in stroke is underscored by the ability ofFactor IXai to reduce microvascular thrombosis (both platelet and fibrinaccumulation are reduced, with an attendant increase in postischemicCBF) and to improve stroke outcome. These potent antithrombotic actionsof Factor IXai are likely to be clinically significant in the setting ofstroke, because Factor IXai not only reduces infarct volumes in adose-dependent manner, but it does so even when given after the onset ofstroke. In addition, at clinically relevant doses, treatment with FactorIXai does not cause an increase in ICH, making selective inhibition ofFactor IXa/VIIIa/X activation complex assembly with Factor IXai anattractive target for stroke therapy in humans.

There are a number of reasons why targetted anticoagulant strategiesmight be an attractive alternative to the current use of thrombolyticagents in the management of acute stroke, because of their checkeredsuccess in clinical trials. Theoretically, an ideal treatment for acutestroke would prevent the formation or induce dissolution of thefibrin-platelet mesh that causes microvascular thrombosis in theischemic zone without increasing the risk of intracerebral hemorrhage.However, thrombolytic agents which have been studied in clinical trialsof acute stroke have consistently increased the risk of intracerebralhemorrhage ¹⁻⁴. Streptokinase, given in the first several (<6) hoursfollowing stroke onset, was associated with an increased rate ofhemorrhagic transformation (up to 67%); although here was increasedearly mortality, surviving patients suffered less residual disability.Administration of tissue-type plasminogen activator (tPA) within 7 hours(particularly within 3 hours) of stroke onset resulted in increasedearly mortality and increased rates of hemorrhagic conversion (between7-20%), although survivors demonstrated less residual disability. Inorder to develop improved anticoagulant or thrombolytic therapies,several animal models of stroke have been examined. These modelsgenerally consist of the administration of clotted blood into theinternal carotid artery followed by administration of a thrombolyticagent. In rats, tPA administration within 2 hours of stroke improvedcerebral blood flow and reduced infarct size by up to 77%^(14,15). In asimilar rabbit embolic stroke model, tPA was effective at restoringblood flow and reducing infarct size, with occasional appearance ofintracerebral hemorrhage^(16,17). However, although there are advantagesto immediate clot dissolution, these studies (as well as the clinicaltrials of thrombolyticv agents) indicate that there is an attendantincreased risk of intracerebral hemorrhage with this therapeuticapproach.

Because of the usually precipitous onset of ischemic stroke, therapy hasbeen targetted primarily towards lysing the majorfibrinous/atheroembolic debris which occludes a major vascular tributaryto the brain. However, as the current work demonstrates there is animportant component of microvascular thrombosis which occurs downstreamfrom the site of original occlusion, which is likely to be ofconsiderable pathophysiological significance for post-ischemichypoperfusion (no-reflow) and cerebral injury in evolving stroke. Thisdata is in excellent agreement with that which has been previouslyreported, in which microthrombi have been topographically localized tothe ischemic region in fresh brain infarcts¹⁸. The use of an agent whichinhibits assembly of the Factor IXa/VIIIa/X activation complexrepresents a novel approach to limiting thrombosis which occurs withinmicrovascular lumena, without impairing extravascular hemostasis, themaintenance of which may be critical for preventing ICH. In the currentstudies, treatment with Factor IXai reduces microvascular platelet andfibrin accumulation, improves postischemic cerebral blood flow, andreduces cerebral infarct volumes in the setting of stroke withoutincreasing ICH.

The potency of Factor IXai as an anticoagulant agent stems from theintegral role of activated Factor IX in the coagulation cascade. Notonly does a strategy of Factor IXa blockade appear to be effective inthe setting of stroke, but it also appears to be effective at preventingprogressive coronary artery occlusion induced following the initialapplication of electric current to the left circumflex coronary arteryin dogs¹³. As in those studies, in which Factor IXai did not prolong theactivated partial thromboplastin time (APTT) (224), in the murine model,administration of Factor IXai at the therapeutically effective dose of300 μg/kg similarly did not significantly alter either the protime orthe APTT (13.4±0.7 and 79.9±8.9 vs 12.1±0.7 and 70.6±8.9 for PT andAPTT, of IXai-treated (n=7) and vehicle treated (n=4) mice,respectively, P=NS).

The data which demonstrates that IXai given after the onset of stroke iseffective leads to another interesting hypothesis, that the formation ofthrombus represents a dynamic equilibrium between the processes ofongoing thrombosis and ongoing fibrinolysis. Even under normal(nonischemic) settings, this dynamic equilibrium has been shown to occurin man¹⁹. The data in the current studies, which show that Factor IXaiis effective even when administered after the onset of stroke, suggeststhat this strategy restores the dynamic equilibrium, which is shiftedafter cerebral ischemia to favor thrombosis, back towards a morequiescent (antithrombotic) vascular wall phenotype.

As a final consideration, even if thrombolysis successfully removes themajor occluding thrombus, and/or anticoagulant strategies are effectiveto limit progressive microcirculatory thrombosis, blood flow usuallyfails to return to pre-ischemic levels. This is exemplified by data inthe current study, in which although CBF is considerably improved byFactor IXai (which limits fibrin/platelet accumulation), CBF still doesnot return to preischemic levels. This data supports the existence ofmultiple effector mechanisms for postischemic cerebral hypoperfusion,including postischemic neutrophil accumulation and consequentmicrovascular plugging, with P-selectin and ICAM-1 expression bycerebral microvascular endothelial cells being particularly germane inthis regard^(5,6). When looked at from the perspective of leukocyteadhesion receptor expression, even when these adhesion receptors areabsent, CBF levels are improved following stroke compared with controlsbut do not return to preischemic levels. Taken together, these datasuggests that microvascular thrombosis and leukocyte adhesion togethercontribute to postischemic cerebral hypoperfusion.

In summary, administration of a competitive inhibitor of Factor IXa,active-site blocked Factor IXa, represents a novel therapy for thetreatment of stroke. This therapy not only reduces microcirculatorythrombosis, improves postischemic cerebral blood flow, and reducescerebral tissue injury following stroke, but it can do so even if givenafter the onset of cerebral ischemia and without increasing the risk ofICH. This combination of beneficial properties and relatively lowdownside risk of hemorrhagic transformation makes this an extremelyattractive approach for further testing and potential clinical trials inhuman stroke.

References.

1. The National Institute of Neurological Disorders and Stroke rt-PAStroke Study Group: Tissue plasminogen activator for acute ischemicstroke. New Engl J Med 1995;333:1581-1587

2. Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von Kummer R, BoysenG, Bluhmki E, Hoxter G, Mahagne M H, Hennerici M, for the ECASS StudyGroup: Intravenous thrombolysis with recombinant tissue plasminogenactivator for acute hemispheric stroke. J A M A 1995;274(13):1017-1025

3. del Zoppo G J: Acute stroke—on the threshold of a therapy. N Engl JMed 1995;333(13):1632-1633

4. Hommel M, Cornu C, Boutitie F, Boissel J P, The MultiCenter AcuteStroke Trial—Europe Study Group: Thrombolytic therapy with streptokinasein acute ischemic stroke. N Engl J Med 1996;335:145-150

5. Connolly E S Jr, Winfree C J, Springer T A, Naka Y, Liao H, Yan S D,Stern D M, Solomon R A, Gutierrez-Ramos J-C, Pinsky D J: Cerebralprotection in homozygous null ICAM-1 mice after middle cerebral arteryocclusion. Role of neutrophil adhesion in the pathogenesis of stroke. JClin Invest 1996;97:209-216

6. Exacerbation of cerebral injury in mice which express the P-selectingene: identification of P-selectin blockade as a new target for thetreatment of stroke. Example 10 Hereinabove

7. Connolly E S Jr, Winfree C J, Stern D M, Solomon R A, Pinsky D J:Procedural and strain-related variables significantly affect outcome ina murine model of focal cerebral ischemia. Neurosurg 1996;38(3):523-532

8. Naka, Y, Chowdhury N C, Liao H, Roy D K, Oz, M C, Micheler R E,Pinsky D J: Enhanced preservation of orthotopically transplanted ratlungs by nitroglycerin but not hydralazine: requirement for graftvascular homeostasis beyond harvest vasodilation. Circ Res1995;76:900-906

9. Lawson C A Yan S-D, Yan S-F, Liao H, Chen, G, Sobel J, Kisiel W,Stern D M, Pinsky D J: Moncytes and tissue factor promote thrombosis ina murine model of oxygen deprivation. Journal of Clinical Investigation1997;99:1729-1738

10Lahiri B, Koehn J A, Canfield R E, Birken S, Lewis J; Development ofan immunoassay for the COOH-terminal region of the gamma chains if humanfibrin. Thromb Res 1981;23:103-112

11. Use of a spectrophotometric hemoglobin assay to objectively quantifyintracerebral hemorrhage in mice. Example 12 Hereinabove

12. Choudhri T F, Hoh B L, Solomon R A, Connolly E S, Pinsky D J:Spectrophotometric hemoglobin assay: A new method to quantifyexperimental murine intracerebral hemorrhage and its potentiation bytissue plasminogen activator. Annual Meeting Joint Section onCerebrovascular Surgery 1997

13. Benedict C R, Ryan J, Wolitzky B, Ramos R, Gerlach M, Tijburg P,Stern D: Active site-blocked Factor IXa prevents intravascular thrombusformation in the coronary vasculature without inhibiting extravascularcoagulation in a canine thrombosis model. J Clin Invest1991;88:1760-1765

14. Papadopoulos S M, Chandler W F, Salamat M S, Topol E J, SackellaresJ C: Recombinant human tissue-type plasminogen activator therapy inacute thromboembolic stroke. J Neurosurg 1987;67:394-398

15. Overgaard K, Sereghy T, Pedersen H, Boysen G: Neuroprotection withNBQX and thrombolysis with rt-PA in rat embolic stroke. Neurol Res1993;15:344—349

16. Carter L P, Guthkelch A N, Orozco J, Temeltas O: Influence of tissueplasminogen activator and heparin on cerebral ischemia in a rabbitmodel. Stroke 1992;23:883-888

17. Phillips D a, Fisher M, Davis M A, Smith T W, Pang R H L: Delayedtreatment with a t-PA analogue and streptokinase in a rabbit embolicstroke model. Stroke 1990;21:602-605

18. Heye N, Paetzold C, Steinberg R, Cervos-Navarro J: The topography ofmicrothrombi in ischemic brain infarct. Acta Neurologica Scandinavica1992;86:450-454

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What is claimed is:
 1. A method for treating an ischemic disorder in a subject which comprises administering to the subject a pharmaceutically acceptable form of inactivated Factor IX in a sufficient amount over a sufficient period of time to inhibit coagulation so as to treat the ischemic disorder in the subject, wherein the inactivated Factor IX migrates identically with Factor IXa on SDS-PAGE.
 2. The method of claim 1, wherein the amount comprises from about 75 μg/kg to about 550 μg/kg.
 3. The method of claim 1, wherein the amount comprises 300 μg/kg.
 4. The method of claim 1, wherein the pharmaceutically acceptable form comprises inactivated Factor IX and a pharmaceutically acceptable carrier.
 5. The method of claim 4, wherein the carrier comprises an aerosol intravenous, oral or topical carrier.
 6. The method of claim 1, wherein the subject is a mammal.
 7. The method of claim 6, wherein the subject is a human.
 8. The method of claim 1, wherein the ischemic disorder comprises a peripheral vascular disorder, a pulmonary embolus, a venous thrombosis, a myocardial infarction, a transient ischemic attack, unstable angina, a reversible ischemic neurological deficit, sickle cell anemia or a stroke disorder.
 9. The method of claim 1, wherein the subject is undergoing heart surgery, lung surgery, spinal surgery, brain surgery, vascular surgery, abdominal surgery, or organ transplantation surgery.
 10. The method of claim 9, wherein the organ transplantation surgery comprises heart, lung, pancreas or liver transplantation.
 11. The method of claim 1, further comprising administering a thrombolytic agent to the subject.
 12. The method of claim 11, wherein the thrombolytic agent is tissue plasminogen activator.
 13. The method of claim 1, wherein the amount comprises from about 75 μg/kg to about 1200 μg/kg.
 14. The method of claim 1, wherein the amount comprises about 600 μg/kg.
 15. The method of claim 1, wherein the amount comprises about 300 μg/kg.
 16. The method of claim 1, wherein the inactivated Factor IX is heat inactivated.
 17. The method of claim 1, wherein the inactivated Factor IX is chemically inactivated.
 18. The method of claim 1, wherein the inactivated Factor IX is inactivated via the active site being blocked.
 19. The method of claim 1, wherein the inactivated Factor IX is inactivated via dansylation of the active site. 